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Thyroid hormone increases the Ca2+-ATPase activity of the sarcoplasmic reticulum (SR) in skeletal muscle, thereby increasing the energy-turnover associated with Ca2+-cycling during contraction and rest. The fast-muscle isoform of the Ca2+-ATPase (SERCA1) and the slow-muscle isoform (SERCA2a), are encoded by two genes that are transcriptionally regulated by T3. The SERCA1 isoform can be expressed to considerably higher levels than the SERCA2a isoform. The stimulation of transcription of the SERCA1 gene by T3 is mediated by two thyroid hormone response elements, located in the promoter of this gene. The intracellular [Ca2+] can modulate the effect of T3. The increase in SR Ca2+-ATPase activity seen when T3-levels rise above normal, results from the induction of SERCA1 expression in slow muscle fibers. Concomitant high levels of Ca2+-ATPase activity are associated with down-regulation of SERCA2a expression in these fibers. The observed T3-dependent increase in SERCAI expression and associated Ca2+ATPase activity will increase the overall metabolic rate of the organism significantly under normal conditions, because of the high average level of contractile activity of slow fibers. Given the rise in serum T3-levels during prolonged cold exposure, these data suggest that fiber-specific stimulation of SERCA1 expression contributes to the thermogenic response in non-shivering thermogenesis. This mechanism may be particularly relevant in larger mammals, which have a relatively high percentage of slow fibers in skeletal muscle, and which need to rely on tissues other than brown fat for the generation of extra heat.

We have previously demonstrated an interaction between the major determinants of skeletal muscle phenotype by showing that continuous contractile activity represses the thyroid hormone (3,3', 5-tri-iodothyronine; T3)-dependent transcriptional activity of fast-type sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic of the fast phenotype. Both the free cytosolic Ca2+ concentration ([Ca2+]i) and the myogenic determination factors MyoD and myogenin have been implicated as mediators of the effect of contractile activity on skeletal muscle phenotype. Using L6 cells we have shown that an increase in the steady-state [Ca2+]i above the resting level of 120 nM indeed can mimic the effect of contractile activity on T3-dependent SERCA1 expression. We now show that the repressing effect of increased [Ca2+]i on T3-dependent SERCA1 expression in L6 cells is exerted at a pre-translational level and is accompanied by increased myogenin mRNA expression. Myogenin overexpression in these cells revealed that increased expression of myogenin alone strongly decreases the T3-dependent stimulation of SERCA1 promoter activity. These results suggest a pathway for the regulation of skeletal muscle phenotype in which [Ca2+]i mediates the effect of contractile activity by regulating the expression of myogenin, which in turn interferes with transcriptional regulation by T3.

Decreased expression of the cardiac slow-twitch sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2), a major determinant of Ca2+ homeostasis, contributes to the abnormal intracellular Ca2+ handling in the failing heart. We investigated the contractility dependence of the effects of norepinephrine (NE) and thyroid hormone (T3) on SERCA2 expression in cultured neonatal heart cells under serum-free conditions. NE and T3 are associated with pathological and physiological forms of hypertrophy, respectively, whereas both hormones increase contractility. In contracting cultures, T3 increased SERCA2 protein and mRNA levels by 35 and 110%, respectively. The same stimulatory effects of T3 on SERCA2 expression were found in contraction-arrested cells. In contracting cultures, NE induced a decrease of SERCA2 protein and mRNA levels by 40 and 60%, respectively. In contrast, SERCA2 protein and mRNA levels were not decreased by NE in contraction-arrested cells, indicating that contractility is a prerequisite for the negative influence of NE on SERCA2 expression. Electrical stimulation at a fixed frequency in the presence and absence of NE demonstrated that the NE-induced increase in contraction frequency is unlikely to account for the decreased SERCA2 expression induced by NE. The results suggest that the effect of contractility on SERCA2 expression depends on the signal transduction pathways that are activated by NE and T3.

Chronic low-frequency contraction of skeletal muscle, either induced by a slow motor nerve or through direct electrical stimulation, generally induces expression of proteins associated with the slow phenotype, while repressing the corresponding fast isoforms. Contractions thereby counteract the primarily transcriptional effect of thyroid hormone (T3) which results in the selective induction and stimulation of expression of fast isoforms. We studied the regulation of expression of the fast-type sarcoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic component of the fast phenotype. Previous work suggested that reduction of SERCA1 expression by contractile activity might result from interference with the T3-dependent transcriptional stimulation of the SERCA1 gene. The present study was set up to test this unexpected mode of action of contractile activity. We show that electrical stimulation of C2C12 mouse myotubes, which results in synchronous contractions at the imposed frequency, reduces basal but virtually abolishes T3-dependent SERCA1 expression. T3-dependent expression of a reporter gene driven by the SERCA1 promoter was similarly affected by electrical stimulation. This is the first demonstration that the counteracting effects on muscle gene expression of electrically induced contractions and T3 may interact at the transcriptional level.

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.

The aim of this study was to investigate the mechanism(s) underlying the thyroid-hormone (L-tri-iodothyronine, T3)-induced elevation of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase (SERCA1) levels in L6 myotubes and the potentiating effect of insulin-like growth factor-I (IGF-I) [Muller, van Hardeveld, Simonides and van Rijn (1991) Biochem. J. 275, 35-40]. T3 increased the SERCA1 protein level (per microgram of DNA) by 160%. The concomitant increase in the SERCA1 mRNA level was somewhat higher (240%). IGF-I also increased SERCA1 protein (110%) and mRNA levels (50%), whereas IGF-I + T3 increased SERCA1 protein and mRNA levels by 410% and 380% respectively. These SERCA1 mRNA analyses show that the more-than-additive action of T3 and IGF-I on SERCA1 expression is, at least in part, pre-translational in nature. Further studies showed that the half-life of SERCA1 protein in L6 cells (17.5 h) was not altered by T3. In contrast, IGF-I prolonged the half-life of SERCA1 protein 1.5-1.9-fold, which may contribute to the disproportional increase in SERCA1 protein content compared with mRNA by IGF-I. Measurements of SERCA1 mRNA half-life (as determined by actinomycin D chase) showed no difference from the control values (15.5 h) in the presence of T3 or IGF-I alone. When T3 and IGF-I were both present, the SERCA1 mRNA half-life was prolonged 2-fold. No significant effects of T3 and IGF-I were observed on the half-life of total protein (37.4 h) and total RNA (37.0 h). The absence of an effect of T3 on SERCA1 protein and mRNA stability, when it was present alone, suggested transcriptional regulation, which was confirmed by nuclear run-on experiments, showing a 3-fold increase in transcription frequency of the SERCA1 gene by T3. We conclude that the synergistic stimulating effects of T3 and IGF-I on SERCA1 expression are the result of both transcriptional and post-transcriptional regulation. T3 acts primarily at the transcriptional level by increasing the transcription frequency of the SERCA1 gene, whereas IGF-I seems to act predominantly at post-transcriptional levels by enhancing SERCA1 protein and mRNA stability, the latter, however, only in the presence of T3.

Thyroid hormone increased the percentage of fibers expressing fast-type sarcoplasmic reticulum Ca(2+)-ATPase in the slow rat soleus muscle from 17% in the hypothyroid to 100% in the hyperthyroid state. This was accompanied by a 12-fold increase in the fast-type Ca(2+)-ATPase protein content of soleus muscle homogenates, suggesting that also the amount of this protein per muscle fiber was increased. In contrast to the fast-type isoform, a decrease in the percentage of fibers expressing slow-type Ca(2+)-ATPase from 100% to 70% was observed in the transition from the hypothyroid to the hyperthyroid state. Slow-type Ca(2+)-ATPase protein levels in muscle homogenates however did not decrease on the same trajectory, but were even elevated in the euthyroid state. In the fast extensor digitorum longus muscle qualitatively similar changes in Ca(2+)-ATPase isoform expression were observed. The results suggest a dual action of thyroid hormone: 1. increasing slow-type Ca(2+)-ATPase expression in individual fibers 2. decreasing the fraction of slow-type Ca(2+)-ATPase expressing fibers.

Net synthesis of the fast-type sarcoplasmic reticulum (SR) Ca2(+)-ATPase was studied in the muscle cell line L6AM using an immunochemical assay (e.l.i.s.a.). In addition, Ca2+ uptake by SR was monitored in muscle cell homogenates by a method employing the fluorescent Ca2+ indicator fura-2. Measurements were done both in differentiating myoblasts and in myotubes. Ca2(+)-ATPase levels were low (1 pmol/mg of protein) in undifferentiated myoblasts (controls) and only doubled over a period of 8 days in the absence of thyroid hormone (L-triiodothyronine; T3). This corresponded to a similar increase in Ca2+ uptake activity. Only half of the myoblasts fused under these conditions. Fusion was not increased in the presence of T3 (5 nM), but Ca2(+)-ATPase levels increased 4-fold and the Ca2+ uptake activity doubled compared with controls. In contrast, insulin-like growth factor-I (IGF-I) induced almost complete myotube formation (greater than 90% fusion), but only slightly stimulated (50%) net Ca2(+)-ATPase synthesis above control levels. However, the doubling of the Ca2+ uptake stimulation by IGF-I was comparable with that caused by T3. The effects of T3 plus IGF-I on Ca2(+)-ATPase levels and Ca2+ uptake activity were more than additive. Furthermore, the temporal relationship between the induction of Ca2(+)-ATPase net synthesis and Ca2+ uptake activity was identical with the two hormones. Qualitatively similar results were obtained when T3 and IGF-I were added to maximally fused cell cultures. The enhanced effect of T3 on Ca2(+)-ATPase net synthesis and Ca2+ uptake activity in the presence of IGF-I cannot therefore be explained by an increased myotube formation stimulated by the latter. In both differentiating myoblasts and myotubes the effect of T3 was more prominent on Ca2(+)-ATPase net synthesis than on Ca2+ uptake activity, whereas in myotubes the opposite was observed for IGF-I. This could imply complementary actions of the two agents in the development of a functional SR.

Basal plasma levels for adrenalin (A), noradrenalin (NA), L-triiodothyronine (T3), and L-thyroxine (T4) were determined in rats with a chronically inserted catheter. The experiments described in this report were started 3 days after the surgical procedure when T3 and T4 levels had returned to normal. Basal levels for the catecholamines were reached already 4 h after the operation. The T3/T4 ratio in plasma was significantly increased after 3, 7, and 14 days in rats kept at 4 degrees C and the same holds for the iodide in the 24-h urine after 7 and 14 days at 4 degrees C. The venous NA plasma concentration was increased 6- to 12-fold during the same period of exposure to cold, whereas the A concentration remained at the basal level. During infusion of NA at 23 degrees C the T3/T4 ratio in plasma was significantly increased after 7 days compared to pair-fed controls, and the same holds for the iodide excretion in the 24-h urine. This paper presents further evidence for a role of the sympathetic nervous system on T4 metabolism in rats at resting conditions.

Rats were made hyperthyroid by repeated sc injections of L-thyroxine (T4) in a two-week period. At several time-points after the last injection, the triiodothyronine (T3) and T4 concentrations in plasma and red and white skeletal muscle were determined by a radioimmunoassay. This was done to investigate the relationship between the change in thyroid-hormone concentration in plasma and muscle and to see whether this change was similar in both muscle types. The results show that: 1. Information about the T3 and T4 concentrations in the muscles of the hyperthyroid rats can only be obtained by direct measurement in the muscles and cannot be gathered from the plasma-T3 concentration at any time-point or from below-normal plasma-T4 concentrations. 2. In hyperthyroid rats T4 and T3 are cleared more rapidly from the plasma than from the skeletal muscle. 3. A proportionally higher T3 concentrations is present in the red compared with the white skeletal muscle of hyperthyroid rats.

Rats were exposed to cold (4 degrees C) for 1 and 4 weeks. The T4 plasma concentrations initially declined (24.5 +/- 7.7 nmol/l) after 1 week but returned to normal levels after 4 weeks (52.9 +/- 14.2 nmol/l). The T3 concentrations were elevated after both 1 and 4 weeks at 4 degrees C (1.31 +/- 0.21 and 1.38 +/- 0.12 nmol/l, respectively). Control values (23 degrees C) for T4 were 42.6 +/- 10.3 and for T3 1.11 +/- 0.13 nmol/l. Addition of 0.015 g KI/l to the drinking water prevented the T4 decrease in plasma after 1 week of cold exposure. No effect of iodide was observed at 23 degrees C. The suppletion of KI did not change pattern of T3 increase after cold exposure. After 4 weeks of cold exposure the T4 levels of the iodide-supplemented animals did not differ from the non-sulemented group. No evidence was found that increased food intake is a contributory factor in the development leading to increased T3 plasma levels during cold exposure.

T3 and T4 concentrations were determined in plasma and red and white skeletal muscle of the rat. Because of the small tissue samples (+/- 300 mg), the ultra-sensitive Wick radioimmunoassay (RIA) for serum was adapted for determination in ethanol extracts. The dilution curves of the plasma and tissue extracts showed excellent parallelism with the standard curves for both T3 and T4. The mean T4 level found in female rats (n = 6) was 22.6 +/- 5.2 ng/ml in plasma and did not differ significantly between red (1.85 +/- 0.28 ng/g) and white (1.90 +/- 0.25 ng/g) skeletal muscle. The mean T3 level in 11 normal female rats was 0.629 +/- 0.0098 ng/ml in the plasma and was significantly higher in the red muscle (2.07 +/- 0.26 ng/g) than in the white muscle (1.65 +/- 0.20 ng/g). The higher T3 levels found in the red muscle as compared with the white muscle may help to elucidate the different responsiveness of these muscle types observed in altered thyroid states.

The effect of sympathetic activity on T4 and T3 levels in cold-exposed rats was investigated. Administration of the highest dose of propranolol (2 mg/100 g b.w.) twice daily during 4 days decreased T4 and T3 concentrations in plasma of rats living at 23 degrees C (T4 from 46.4 +/- 2.6 to 25.8 +/- 5.3 nmol/l and T3 from 1.08 +/- 0.6 to 0.82 +/- 0.12 nmol/l). No significant effect on T4 and T3 levels (49.0 +/- 11.6 and 1.48 +/- 0.16 n/mol, respectively) after the administration of the same dose regimen of propranolol was observed in rats exposed to cold for 4 weeks. T4 and T3 levels in rats exposed to cold for 4 weeks were not significantly altered 1 week after sympathectomy, while remaining in the cold. However, chemical sympathectomy before cold exposure delayed the cold induced T3 elevation occurring during the first week of cold exposure (controls: from 1.16 +/- 0.19 to 1.44 +/- 0.29 nmol/l; sympathectomized rats: from 1.07 +/- 0.12 to 1.17 +/- 0.22 nmol/l). After 2 weeks of cold exposure the T3 levels of controls and sympathectomized rats were not significantly different (controls: 1.45 +/- 0.12 nmol/l, sympathectomized rats: 1.38 +/- 0.15 nmol/l). No effect of sympathectomy was observed on T4 levels. These experiments show that the role of sympathetic activity in increasing T3 is not clear during cold exposure. They provide some evidence that sympathetic activity may play a role in the initiation of the process leading to increased T3 plasma levels during cold exposure.

1. A modified method for the determination of thyroid hormone levels in muscle is described. 2. The levels of T4 and T3 in skeletal muscle tissue of rats lie in the same range. 3. The level of T4 in plasma is about a hundred times higher than that of T3. 4. To remove plasma hormones from the tissue in which hormone levels are to be determined, the animals should be perfused with saline before tissue samples are taken.