Mechanism of thyroid-hormone regulated expression of the SERCA genes in skeletal muscle: implications for thermogenesis.

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.

PKC-dependent preconditioning with norepinephrine protects sarcoplasmic reticulum function in rat trabeculae following metabolic inhibition.

The authors have previously shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition, and improves contractile function during a subsequent recovery period. The present study investigated: (i) whether protection of sarcoplasmic reticulum (SR) function during metabolic inhibition (MI) is involved in the preconditioning-like effect of NE-pretreatment, and (ii) whether or not this process is PKC-dependent. A 15 min preincubation period was used with 1 micromol/l exogenous NE to precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 40 min of MI in 2 mmol/l NaCN containing Tyrode (gassed with 95% O2/5% CO2; pH 7.4, 24 degrees C) without glucose at 1-Hz stimulation frequency. Contractile recovery was studied during a subsequent 60 min recovery period (RP) in glucose containing Tyrode at 0.2 Hz. Force and intracellular free calcium ([Ca2+]ii) were monitored throughout the experimental protocol. Pretreatment of trabeculae with NE (group NE) substantially diminished the Ca2+ rise from the onset of rigor development during MI, compared to preparations which were pretreated with NE, in the presence of specific PKC blocker chelerythrine (2 micromol/l; group NE+CHEL). After 40 min of MI, resting [Ca2+]i in group NE and NE+CHEL was increased to 0.50+/-0.03 and 2.08+/-0.20 micromol/l, respectively (P<0.05), whereas total intracellular ATP levels were similar in both groups (approximately 0.20 micromol/g dry wt). This corresponded with an increase in active force development (119%) and a decrease in twitch force relaxation time (77%) during subsequent RP in group NE, compared to pre-MI values of the same group. In contrast, a significant decrease in force recovery (54%) and an increase in twitch force relaxation time (123%) was observed in group NE+CHEL. Values for [Ca2+]i, contractile recovery, and twitch force relaxation time in untreated controls as well as CHEL preparations corresponded to those measured in the NE+CHEL group. Rapid cooling contractures (RCCs), which provide information on both SR-Ca2+ loading and Ca2+ re-uptake activity, revealed a 2-fold higher SR Ca2+ content during RP in group NE compared to controls and group NE+CHEL. In addition, kinetic analysis of the RCC rewarming spike (RWS) showed that this was accompanied by greater than a 28% increase in the maximum rate of RWS relaxation (-dF/dt/rws) in group NE compared to group NE+CHEL. The change of -dF/dt/rws in the NE group during RP following MI persisted after SR Ca2+-release channel blockade by ryanodine treatment (100 micromol/l), which suggests involvement of NE-induced, PKC-dependent protection of SR Ca2+-ATPase activity. The results of the present study point to an inverse relationship between the Ca2+ rise during MI and SR functioning, in which PKC appears to play a key role. It is concluded that the preconditioning-like effect of NE-pretreatment on contractile recovery is at least partly mediated by protection of SR function.

Differential effects of norepinephrine on contractile recovery of rat trabeculae following metabolic inhibition.

We have recently shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition (MI) with NaCN (2 mmol/l), and improves contractile recovery during a subsequent recovery period (RP). In the present study, we investigated the effects of the continuous presence of NE (1 micro mol/l), i.e. before, during and after MI, on Ca2+ homeostasis maintenance and contractile recovery in the same model at 24 degrees C. In addition, we tested the effects of NE when only present in the rigor period during MI. The continuous presence of NE both before (30 min) and during MI (120 min)+RP (60 min) (group NE-I) significantly increased the proportion of trabeculae that resumed to contract during RP from 46+/-4% (mean+/-s.e.m.) in controls to 82+/-8%. The Ca2+ rise at the end of MI in failing control trabeculae (1.85+/-0.04 micro mol/l) was more than doubled compared to recovering control preparations (0.78+/-0.02 micro mol/l). However, the time-course of the Ca2+ rise during MI in recovering and failing NE-I preparations was similar, and eventually of the same magnitude as observed in failing control preparations (1.6+/-0. 02 and 1.85+/-0.07 micro mol/l, respectively). In contrast, when NE was present only in the rigor period during MI (group NE-II) the proportion of recovering preparations decreased significantly to 27+/-9%. Similar to the control group, recovering and failing preparations in group NE-II could be distinguished by a differential course in the Ca2+ rise during MI. The results show that when NE is present both before and during MI+RP, (i) recovery probability following MI is still improved, in spite of the deleterious effect on contractile recovery of the presence of NE in the rigor during MI, and (ii) there is no relationship between the magnitude of Ca2+ overload during MI and recovery probability during RP.

Cross-talk between transcriptional regulation by thyroid hormone and myogenin: new aspects of the Ca2+-dependent expression of the fast-type sarcoplasmic reticulum Ca2+-ATPase.

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.

Norepinephrine pretreatment attenuates Ca2+ overloading in rat trabeculae during subsequent metabolic inhibition: improved contractile recovery via an alpha 1-adrenergic, PKC-dependent signaling mechanism.

The present study was designed in order to investigate more precisely the role of calcium homeostasis maintenance in protein kinase C (PKC) mediated preconditioning. We used a 15 min pre-incubation period, with 1 mumol/l exogenous norepinephrine (NE) to pharmacologically precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 120 min of metabolic inhibition (MI, in 2 mmol/l CN- containing Tyrode without glucose at 1 Hz stimulation frequency). Contractile recovery was studied during a subsequent 60 min recovery period (RP, in glucose containing Tyrode at 0.2 Hz). Tyrode was gassed with 95%, O2/ 5% CO2 and kept at a constant temperature of 24 degrees C. Force and intracellular free calcium ([Ca2+]i) were monitored throughout the experimental protocol; [Ca2+]i was measured using fura-2. Pretreatment with NE (group NE-I) significantly increased the fraction of trabeculae that resumed to contract during RP, from 36 +/- 13% (mean +/- S.E.M.) in controls to 82 +/- 10% (P < 0.05). In correspondence with this, NE-pretreatment increased the proportion of trabeculae in which the Ca2+ rise from the onset of rigor development during MI was attenuated. After 40 min of MI [Ca2+]i in the failing control, as well as failing group NE-I, trabeculae (1.08 +/- 0.20 and 1.51 +/- 0.26 mumol/l, respectively) was increased significantly compared to the mean value registered in the recovering preparations of these groups (0.34 +/- 0.04 mumol/l: P < 0.05). Specific inhibition of PKC with 2 mumol/l chelerythrine (group NE-IV) almost completely blocked the protection induced by NE-pretreatment, including its protective action against Ca2+ overload, i.e. the fraction of trabeculae that resumed to contract during RP returned to untreated control level (46 +/- 11%: P < 0.05 v group NE-I). Also in this case [Ca2+]i in the failing group NE-IV trabeculae after 40 min of MI was increased substantially, compared to the value measured in the recovering preparations (4.75 +/- 1.00 and 0.60 +/- 0.08 mumol/ l, respectively). The relative importance of both alpha-adrenergic and beta-adrenergic receptor pathways in this preconditioning-like effect of NE-pretreatment, was investigated using specific blockers. The results point to an alpha 1-adrenergic receptor mediated signaling mechanism, which enhances PKC-dependent control of [Ca2+]i from the onset of rigor development during MI.

Modulation of SERCA2 expression by thyroid hormone and norepinephrine in cardiocytes: role of contractility.

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.

Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene.

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.

The effect of hyperosmolality on the rate of heat production of quiescent trabeculae isolated from the rat heart.

We have measured the rate of heat production of isolated, quiescent, right ventricular trabeculae of the rat under isosmotic and hyperosmotic conditions, using a microcalorimetric technique. In parallel experiments, we measured force production and intracellular calcium concentration ([Ca2+]i). The rate of resting heat production under isosmotic conditions (mean +/- SEM, n = 32) was 100 +/- 7 mW (g dry wt)-1; it increased sigmoidally with osmolality, reaching a peak that was about four times the isosmotic value at about twice normal osmotic pressure. The hyperosmotic thermal response was: (a) abolished by anoxia, (b) attenuated by procaine, (c) insensitive to verapamil, ouabain, and external calcium concentration, and (d) absent in chemically skinned trabeculae bathed in low-Ca2+ "relaxing solution." Active force production was inhibited at all osmolalities above isosmotic. Passive (tonic) force increased to, at most, 15% of the peak active force developed under isosmotic conditions while [Ca2+]i increased, at most, 30% above its isosmotic value. We infer that hyperosmotic stimulation of resting cardiac heat production reflects, in large part, greatly increased activity of the sarcoplasmic reticular Ca2+ ATPase in the face of increased efflux via a procaine-inhibitable Ca(2+)-release channel.

Characterization of the promoter of the rat sarcoplasmic endoplasmic reticulum Ca2+-ATPase 1 gene and analysis of thyroid hormone responsiveness.

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.

Expression of MyoD in cultured primary myotubes is dependent on contractile activity: correlation with phenotype-specific expression of a sarcoplasmic reticulum Ca(2+)-ATPase isoform.

Myogenic determination factors (MDF) have been implicated in the establishment and maintenance of the fast or slow phenotype in skeletal muscle, with MyoD favoring the fast and myogenin favoring the slow phenotype. Accordingly, contractility-induced changes in muscle phenotype should be accompanied by a change in the MyoD/myogenin ratio. Some reports show such changes, but limitations inherent to in vivo studies complicate interpretation of these data. Here we tested whether a relationship can be found between contractility, MDF expression, and the expression of phenotype-specific muscle proteins in a simple in vitro system of cultured primary myotubes. We show that contractions reduce the MyoD/myogenin ratio by specifically repressing MyoD mRNA expression. This is accompanied by a selective repression at a pretranslational level of the expression of fast-type sarcoplasmic reticulum Ca(2+)-ATPase. These in vitro results support a phenotype-determining role of MDFs as a function of contractile activity and show that cultured myotubes can be a useful model for the analysis of the molecular mechanism of such regulation of muscle phenotype.

Fiber-specific regulation of Ca(2+)-ATPase isoform expression by thyroid hormone in rat skeletal muscle.

We studied the effect of thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the expression of sarcoplasmic reticulum (SR) fast- and slow-type Ca(2+)-ATPase isoforms, SERCA1 and SERCA2a, respectively, and total SR Ca(2+)-ATPase activity in rat skeletal muscle. Cross sections and homogenates of soleus and extensor digitorum longus muscles from hypo-, eu-, and hyperthyroid rats were examined, and expression of Ca(2+)-ATPase isoforms in individual fibers was compared with expression of fast (MHC II) and slow (MHC I) myosin heavy chain isoforms. In both muscles, T3 induced a coordinated and full conversion to a fast-twitch phenotype in one-half of the fibers that were slow twitch in the absence of T3. The conversion was partial in the other one-half of the fibers, giving rise to a mixed phenotype. The stimulation by T3 of total SERCA expression in all fibers was reflected by increased SR Ca(2+)-ATPase activity. The time course of the T3-induced changes of SERCA isoform expression was examined 1-14 days after the start of daily T3 treatment of euthyroid rats. SERCA1 expression was stimulated by T3 at a pretranslational level in all fibers. SERCA2a mRNA expression was transiently stimulated and disappeared in a subset of fibers. In these fibers SR Ca(2+)-ATPase activity was high because of high SERCA1 protein levels. These data suggest that the ultimate downregulation of SERCA2a expression, which is always associated with high SR Ca(2+)-ATPase activities, occurs at a pretranslational level.

Histochemistry of sarcoplasmic reticulum Ca-ATPase using dysprosium as capturing reagent.

This report describes the development of a histochemical method for the demonstration of sarcoplasmic reticulum Ca-ATPase activity in cross-sections of skeletal muscle. The demonstration of sarcoplasmic reticulum Ca-ATPase activity is complicated by the fact that capturing reagents for phosphate inhibit the enzyme. We present a minimal model for heavy-metal-phosphate precipitation reactions which gives a theoretical description of the effect of enzyme inhibition on the rate of phosphate precipitation in the section. The model indicates that the choice of capturing reagent is crucial: whether or not ATPase activity can be demonstrated depends mainly on the inhibition constant and the solubility product of the phosphate salt of the capturing reagent (but also on a fairly large number of other factors). All lanthanides tested can be used to demonstrate sarcoplasmic reticulum Ca-ATPase activity, but dysprosium results in the highest staining intensity. This suggests that dysprosium inhibits sarcoplasmic reticulum Ca-ATPase to a lesser degree than the other lanthanides and/or the solubility product of its phosphate salt is smaller. As an example, the method is used to investigate the effect of thyroid hormone on sarcoplasmic reticulum Ca-ATPase activity in individual fibres of the rat soleus muscle.

Exposure of energy-depleted rat trabeculae to low pH improves contractile recovery: role of calcium.

The beneficial effect of low pH during cardiac ischemia on reperfusion injury has often been attributed to its energy-saving effect due to inhibition of contraction. The role of low pH on Ca2+ accumulation and muscle tension was assessed in energy-depleted tissue by changing the pH of the medium from 7.4 to 6.2 at onset of rigor development during metabolic inhibition (MI), i.e., in the energy-depleted phase. Cytosolic free Ca2+ ([Ca2+]i) and intracellular H+ (pHi) were measured in rat trabeculae at 20 degrees C with fura 2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively, and tension was recorded. The preparations were energy depleted by stimulation at 1 Hz in glucose-free Tyrode solution with 2 mM NaCN. Rigor developed within 20 min, indicating energy depletion. Resting [Ca2+]i was followed during 50 min (group I) or 100 min (group II) of rigor, and recovery was followed for 60 min in glucose-containing Tyrode solution at 0.2-Hz stimulation. Resting [Ca2+]i rose within 50 min (group I) but stabilized in the 50- to 100-min period (group II). All preparations from group I (n = 5) resumed contraction in the recovery period but in group II (n = 10) 70% failed to recover, and [Ca2+]i remained elevated compared with those that recovered. An extracellular pH of 6.2, resulting in similar pHi, from onset of rigor development (group III) led to only a modest rise in [Ca2+]i during the 100-min rigor period, and all preparations resumed contraction after approximately 3 min in normal medium. ATP was very low in all groups at the end of MI but was still significantly lower in group II than in groups I and III. A beneficial energy-sparing effect of low pH during the rigor phase can therefore not be excluded. We conclude that 1) the capacity of trabeculae to recover from MI depends on the time period and magnitude of the [Ca2+]i rise in the energy-depleted phase and 2) low pH in energy-depleted trabeculae protects against Ca overload, improving recovery after normalization of perfusion conditions.

Differential regulation of the expression of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase by thyroid hormone and insulin-like growth factor-I in the L6 muscle cell line.

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.

Differential effects of thyroid hormone on the expression of sarcoplasmic reticulum Ca(2+)-ATPase isoforms in rat skeletal muscle fibers.

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.

Thyroid hormone regulates Ca(2+)-ATPase mRNA levels of sarcoplasmic reticulum during neonatal development of fast skeletal muscle.

In gastrocnemius muscle from newborn rats the mRNA for the fast sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform (SERCA1) comprised over 90% of total SR Ca(2+)-ATPase mRNA content and increased 5-fold between day 5 and 20 after birth, whereas in hypothyroid muscle the SERCA1 message level remained constant. Triiodothyronine (T3) treatment of 2-day-old euthyroid rats induced a precocious stimulation of SERCA1 mRNA levels, indicating that T3 is the determining factor in the stimulation of SERCA1 message levels and that this stimulation underlies the previously reported effect of the thyroid status on the neonatal development of SR Ca(2+)-ATPase activity. The low mRNA level for the slow SR Ca(2+)-ATPase isoform (SERCA2) was constant in both euthyroid and hypothyroid muscle development. Nevertheless, T3 treatment of hypothyroid neonates induced a transient stimulation of SERCA2 message levels, indicating that SERCA2 is responsive to higher levels of T3.

Ca2+ homeostasis and fast-type sarcoplasmic reticulum Ca(2+)-ATPase expression in L6 muscle cells. Role of thyroid hormone.

The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblasts with caffeine or high [K+]o. The role of [Ca2+]i in the regulation of Ca(2+)-ATPase levels was investigated by varying [Ca2+]i through exposure of cells to different concentrations of extracellular Ca2+ (0.2-1.8 mM) and ionomycin (0.1-0.25 microM). At subnormal [Ca2+]i (55 nM) the T3-induced net synthesis of Ca(2+)-ATPase was virtually abolished, and at supranormal [Ca2+]i (195 nM) it was greatly depressed. Intermediate stimulation of net Ca(2+)-ATPase synthesis was found at [Ca2+]i of 95 and 165 nM, with an optimum at approx. 125 nM. Similar but less pronounced effects were found for the basal Ca(2+)-ATPase levels. In contracting primary rat myotubes, Ca(2+)-ATPase levels were significantly lower than in tetrodotoxin-arrested myotubes. The same results were obtained in the presence of T3. Since the mean [Ca2+]i in contracting cells is higher than in resting cells, these data agree with those obtained in the L6 cells with ionomycin. A major conclusion of this study is the existence of a [Ca2+]i optimum, near resting levels, for the expression of the fast-type Ca(2+)-ATPase in the L6 muscle cell line.

The elevation of sarcoplasmic reticulum Ca2(+)-ATPase levels by thyroid hormone in the L6 muscle cell line is potentiated by insulin-like growth factor-I.

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.

An assay for sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates.

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.