Quantification of thyroxine and 3,5,3'-triiodo-thyronine in human and animal hearts by a novel liquid chromatography-tandem mass spectrometry method.

Assaying tissue T3 and T4 would provide important information in experimental and clinical investigations. A novel method to determine tissue T3 and T4 by HPLC coupled to mass spectrometry is described. The major difference vs. previously described methods lies in the addition of a derivatization step, that is, to convert T3 and T4 into the corresponding butyl esters. The yield of esterification was Ì´ 100% for T3 and 80% for T4. The assay was linear (r>0.99) in the range of 0.2-50 ng/ml, accuracy was in the order of 70-75%, and the minimum tissue amount needed was in the order of 50 mg, that is, about one order of magnitude lower than observed with the same equipment (AB Sciex API 4000 triple quadrupole mass spectrometer) if derivatization was omitted. The method allowed detection of T3 and T4 in human left ventricle biopsies yielding concentrations of 1.51±0.16 and 5.94±0.63 pmol/g, respectively. In rats treated with different dosages of exogenous T3 or T4, good correlations (r>0.90) between plasma and myocardial T3 and T4 concentrations were observed, although in specific subsets different plasma T4 concentrations were not associated with different tissue content in T4. We conclude that this method could provide a novel insight into the relationship between plasma and tissue thyroid hormone levels.

Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis.

In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.

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.

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.

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.

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.

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.

Thyroid hormone differentially affects mRNA levels of Ca-ATPase isozymes of sarcoplasmic reticulum in fast and slow skeletal muscle.

mRNA levels for the type I and type II isoforms of sarcoplasmic reticulum (SR) Ca-ATPase were determined in soleus (SOL) and extensor digitorum longus (EDL) muscle of euthyroid (normal), hypothyroid, and hyperthyroid rats. Total Ca-ATPase mRNA content of hyperthyroid muscle was 1.5-fold (EDL) and 6-fold (SOL) higher compared to hypothyroid muscle, with corresponding increases in total SR Ca-ATPase activity. EDL contained only type II Ca-ATPase mRNA. In SOL type I mRNA was the major form in hypothyroidism (98%), but the type II mRNA content was stimulated 150-fold by T3, accounting for 50% of the Ca-ATPase mRNA in hyperthyroidism.

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.

Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis.

The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.