Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca^2 +-ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

Abstract

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca^2 + release, and the light fraction enriched in Ca^2 +-ATPase (SERCA), an enzyme responsible for Ca^2 + transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca^2 +- ATPase activity and Ca^2 +-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca^2 +-uptake and Ca^2 +-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.

• Heavy sarcoplasmic reticulum
• light sarcoplasmic reticulum
• SERCA
• hyperthyroidism
• hypothyroidism

Introduction

The sarcoplasmic reticulum (SR) is a membrane network found in skeletal muscle that functions as an intracellular calcium storage site. The SR structure is composed of two morphologically and functionally distinct fractions. The terminal cisternae or heavy SR fraction corresponds to an enlarged portion of SR, which in addition to the Ca^2 +-ATPase contains proteins involved in calcium storage and release such as calsequestrin and the ryanodine Ca^2 + channel. This part of the reticulum is connected to the muscle cell transverse tubule by the calcium channels and dihydropyridine receptors, a complex that plays a key role in the excitation-contraction coupling mechanism [1], [2]. The light SR fraction corresponds to the longitudinal SR membranes that are spread along the myofibrils. This fraction is enriched in Ca^2 +-ATPase and is involved in muscle relaxation [3–7].

The sarcoplasmic reticulum Ca^2 +-ATPases (SERCA) are a family of membrane bound enzymes that drive the transport of calcium from the cytosol to the sarcoplasmic reticulum lumen using ATP hydrolysis as energy source [8–12]. Different genes encode the sarco/endoplasmic reticulum Ca^2 +-ATPase (SERCA) isoforms, but the physiological significance of this isoform diversity is not clear [13–17]. SERCA 1 and SERCA 2a isoforms expression differ depending on skeletal muscle fiber types. While white muscle fibers (fast-twitch) express solely SERCA 1, red muscle fibers (slow-twitch) express both SERCA 1 and SERCA 2a. The SERCA 2b and SERCA 3 genes are expressed in non-muscular tissues such as blood platelets and lymphoid tissue [13–17].

The thyroid hormone 3,5,3′-triiodo L-thyronine (T₃) regulates the expression and function of SERCA proteins, and this effect varies depending on the fiber type [18–25]. In white skeletal muscle, the SERCA 1 isoform content does not seem to be significantly changed by thyroid status. In the red muscle however, high T3 levels promote an over expression of SERCA 1 isoform, which is accompanied by an increase of both the SR rate of calcium transport and ATPase activity [18], [22]. In hypothyroidism, the expression of SERCA 2a is not altered but there is a decrease in SERCA 1 [20], [25].

Most of the studies measuring SERCA expression in either hypo- or hyperthyroid animals were performed in rats and the Western blot analyses were done using either muscle homogenates or immunohistochemical staining of muscle fibers. Using these methodological approaches it is not possible to ascertain whether the changes of SERCA proteins promoted by T3 differ in the longitudinal and terminal cisternae of the sarcoplasmic reticulum.

In this report we aimed to determine whether the action of T3 on muscles involves the subcellular redistribution of SERCA proteins. We isolated the light and heavy fractions of the SR and measured both the expression of the two SERCA isoforms and the Ca^2 +-transport activity in hypo- and hyperthyroid rabbits.

Materials and methods

Light and heavy SR vesicles derived from rabbit skeletal muscle

Hind limb red and white muscles were dissected from white male rabbits based on their macroscopic aspect. This is a procedure adopted in previous reports (22,41). The rabbits were treated in accordance with the published regulations for animal laboratorial use. Vesicles derived from light and heavy sarcoplasmic reticulum were prepared as previous reports with small modifications [3], [5]. Briefly, white (40 g) and red (10 g) muscles were homogenized in 10 mM MOPS/Tris pH 7.0, 10% Sucrose 0.1 mM EDTA (buffer 1) in a blender. Homogenization and all subsequent steps were performed at 4°C. The homogenates were centrifuged for 10 min at 1,600×g. The supernatants (S1) were filtered through four layers of cheesecloth. The pellet was resuspended in buffer 1 and centrifuged for 10 min at 1,600×g. The supernatant of the second centrifugation (S2) was mixed with S1 and centrifuged for 10 min at 5,200×g. The pellet was discarded and the supernatant was re-centrifuged for 30 min at 15,000×g. The supernatant of this centrifugation was collected (S3). The pellet was homogenized in 10 mM MOPS/Tris pH 7.0 and 0.6 M KCl (buffer 2) and centrifuged for 30 min at 20,000×g. The supernatant was discarded ann the pellet was collected (P1). S3 was centrifuged for 40 min at 30,000×g, the supernatant was discarded and the pellet was homogenized with buffer 2. This homogenate was centrifuged for 20 min at 20,000×g, the supernatant was collected (S4) and the pellet was mixed with the P1 and homogenized in a small volume of 50 mM MOPS/Tris pH 7.0 with 0.6 M sucrose and stored at –70°C. These vesicles correspond to the heavy SR fraction. The S4 was centrifuged for 40 min at 30,000×g, the supernatant was discarded and the pellet was homogenized in a small volume of B3 and stored at −70°C. These vesicles correspond to light SR vesicles. Prior to use, vesicles were diluted in a medium containing 50 mM MOPS/Tris buffer, 100 mM KCl, 10 mM P_i and 10 µM CaCl₂.

Hypo- and hyperthyroid animals

Hypothyroid state was induced by oral administration of propylthiouracil (0.08%), in drinking water for 21 days [26]. Hyperthyroidism was induced in rabbits by injection of T4 (100 µg/kg body weight), subcutaneously, for 10 days [16], [18], [22]. After the period of treatment the animals were sacrificed and blood was collected for T4 measurement. Hypo- and hyperthyroidism were confirmed by measurements of body weight and serum T4 (Table I). Serum total T4 was measured by specific radioimmunoassay kits using ¹²⁵I as tracer (T4: DLS −3200 Active, sensitivity of 0.4 µg/dl, inter- and intra assay coefficients of variation varied from 7.1 to 7.4% and 2.9 to 5.1%, respectively, TX, EUA), based on the presence of specific antibodies adhered to the internal surface of propylene tubes. Rabbit hormone free serum was used in the standard curves for total T4. All the procedures were carried out following the recommendations of the kit.

Table I.  Body weight and serum total T4 in control, hyper and hypothyroid rabbits

	Body Weight (kg)
Experimental Groups	Initial	Final	Serum T4 (µg/dl)
Control	2.27±0.13	2.45±0.10	1.73±0.26
HypoT	2.16±0.11	2.43±0.09	<0.40
HyperT	2.47±0.18	2.05±0.19^a	9.33±0.61^b

Hyperthyroidism was induced by T4 administration for 10 days (100 µg/kg b.w., sc) and hypothyroidism was obtained by propylthiouracil (0.08%) administration in drinking water for 21 days. Values in the table are average±SE, n=6. The decrease of body weight observed in hyperthyroid animals is statistically significant. ^aDifference between initial and final body weight, p<0.05; ^bDifference between hyperthyroid and both control and hypothyroid, p<0.001.

Gel electrophoresis and Western blot

Proteins were separated on a 7.5% polyacrylamide (SDS-PAGE) gel according to Laemmli [27]. Electro transfer of protein from the gel to polyvinylidene difluoride (PVDF) membranes was performed for 20 min at 250 mA per gel in 25 mM Tris, 192 mM glycine and 20% methanol using a Mini Trans-Blot cell from Bio-Rad. Membranes were blocked with 3% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. Membranes were then washed and incubated for 1 h with anti-SERCA 1, anti-SERCA 2 monoclonal antibodies at room temperature. The membranes were washed and blots were revealed using an ECL detection kit from Amersham-Pharmacia Biotech, UK. Monoclonal antibodies for SERCA 1 (clone IIH11) and SERCA 2 (clone IID8) were obtained from Affinity BioReagents, Inc. (Brazil). Densitometric analyses were performed using Scion Image 4.02 for windows, downloaded from web site:scioncorp.com.france.fr (NIH). Total homogenates were obtained by muscle centrifugation at 10,000×g and the supernatant containing practically all the cell components were used, except the actomiosin filaments.

Ca^2 + uptake

Trace amounts of ⁴⁵Ca were included in the assay medium. The reaction was arrested by filtering samples of the assay medium through Millipore filters [28]. After filtration, the filters were washed five times with 5 ml of 3 mM La(NO₃)₃ and the radioactivity remaining on the filters was counted using a liquid scintillation counter.

ATPase activity

Enzyme activity was assayed by measuring the release of ³²P_i from [γ-³²P]ATP. The reaction was arrested with trichloroacetic acid (final concentration 5% w/v). The [γ-³²P]ATP not hydrolyzed during the reaction was extracted with activated charcoal as previously described [29]. Two different ATPase activities can be distinguished in sarcoplasmic reticulum vesicles [8–11], [30]. The Mg^2 +-dependent activity requires only Mg^2 + for its activation and is measured in the presence of 2 mM EGTA to remove contaminant Ca^2 + from the medium. The Ca^2 +-dependent ATPase activity, which is correlated with Ca^2 + transport, is determined by subtracting the Mg^2 +-dependent activity from the activity measured in the presence of both Mg^2 + and Ca^2 +. In previous reports, skeletal muscle calcium dependent ATPase has been shown to be fully inhibited by sub micromolar concentrations of thapsigargin [31].

Experimental procedure

All the experiments were performed at 35°C. In a typical experiment the assay media was divided into three samples, which were used for the simultaneous measurement of Ca^2 + uptake and ATP hydrolysis. These measurements were started simultaneously with vesicles to a final concentration of 5 µg protein/ml. NaN₃ (5 mM), an inhibitor of mitochondrial ATP synthase, was added to the assay medium The free Ca^2 + concentration in the medium was calculated as previously described [32], [33].

Statistical analyses

All enzymatic data were analysed by unpaired t-test. Initial and final rabbit body weights were analysed by paired t-test. Serum T4 levels were analysed by one-way analysis of variance followed by Newman–Keuls multiple comparison tests.

Results and discussion

Characterization of light and heavy SR fractions derived from white and red skeletal muscle

Electrophoretic analyses of muscle vesicles revealed a band of 110 kDa, characteristic of SERCA proteins (Figure 1A). In both red and white muscle from control rabbits, the amount of Ca^2 +-ATPases was more conspicuous in the light than in the heavy fraction (Figure 1B). When compared with white muscle, both the light and heavy fractions of red muscle showed more protein bands. Red muscle contains a larger quantity of mitochondria than white muscle, and during the process of preparation of the SR vesicles mitochondria lysis can lead to the formation of sub-mitochondrial particles that cannot be separated from the SR vesicles fraction. Therefore, the sub-mitochondrial particles are one of the possible contaminants prevailing in red muscle. To explore this possibility we measured the ATPase activity of the mitochondrial F_o F₁ ATP synthase (Figure 2) in the two vesicle fractions. For white muscle, a tissue with low mitochondria content, there was little or no mitochondrial ATPase activity in either the light or the heavy SR fraction (Figure 2A), but in red muscle there was a significant sodium azide sensitive mitochondrial ATPase activity, which was more pronounced in the heavy than in the light SR fraction (Figure 2B).

Figure 1.  Electrophoresis of vesicles obtained from red (RM) and white (WM) skeletal muscle. LF refers to light fraction and HF to heavy fraction. A) The amount of protein used in SDS-PAGE gel electrophoresis was 20 µg for red muscle and 15 µg for white muscle. The gel was stained with Coomassie Brilliant Blue. B) Immunodetection was obtained with SERCA 1 and SERCA 2 specific monoclonal antibodies. 7 µg protein of light and heavy SR vesicles from red muscle and 0.5 µg protein of light and heavy SR vesicles from white muscle were used to load the gels.

Figure 2.  ATP Synthase activity of vesicles obtained from white (A) and red (B) skeletal muscle. This activity was calculated by subtracting the ATPase activity without sodium azide from the ATPase activity in the presence of sodium azide. The assay medium composition was 50 mM Tris/HCl buffer (pH 8.0), 1 mM ATP, 2 mM MgCl₂, 5 mM EGTA, 100 mM KCl and 5 mM NaN3. The reaction was performed at 35°C and was started by the addition of vesicles (5 µg protein/ml). The vesicles used in the experiment were derived from light (•) and heavy (○) SR fractions. The figure shows the average±SE of 4 experiments.

In agreement with previous reports [34], [35], we found that the rate of Ca^2 + uptake and the amount of Ca^2 + accumulated at steady state by the heavy SR are smaller than that measured with the light fractions (compare Figure 3 B and D with Figure 3 A and C). In the literature it is well established that caffeine impairs the Ca^2 + accumulation of the heavy fraction by increasing the Ca^2 + release from the vesicles, but has no effect in the light vesicles fraction [7], [36–38]. Accordingly, Figure 3 and Table II show that caffeine impairs Ca^2 + accumulation from SR of both white and red muscle heavy fractions but has little or no effect on the light fraction of the two muscles.

Figure 3.  Effect of caffeine in light (A and C) and heavy SR (B and D) fraction obtained from red and white skeletal muscles. The assay medium composition was 50 mM Mops/Tris buffer (pH 7.0), 1 mM ATP, 2 mM MgCl₂, 0.2 mM CaCl₂, 0.2 mM EGTA, 10 mM Pi, 100 mM KCl, 10 mM caffeine, 5 mM NaN₃ and trace amounts of ⁴⁵Ca^2 +. The reaction was performed at 35°C and was started by the addition of vesicles (5 µg protein/ml). The calculated free Ca^2 + concentration in the medium was 5 µM. Upper panel – red muscle and lower panel – white muscle. Control (○), 10 mM caffeine (•). The figure shows a typical experiment.

Table II.  Effect of caffeine on Ca^2 +-uptake in white and red skeletal muscle.

	White Muscle (µmolCa^2 +/mg protein.min^-1)		Red Muscle (µmolCa^2 +/mg protein.min^-1)
Treatments	Light SR	Heavy SR	Light SR	Heavy SR
Control	2.96±0.41 (4)	1.92±0.19 (9)	0.98±0.13 (3)	0.16±0.03 (7)
10 mM caffeine	2.68±0.35 (4)	1.36±0.20^a (9)	1.02±0.21 (3)	0.10±0.01^a (7)

Assay medium and experimental conditions were as described in Figure 3. Values are mean±SE. The numbers of experiments are in parentheses. ^aDifference between 10 mM caffeine and control, p<0.025.

Hyperthyroidism

There is general agreement that in both rats [18–21], [24] and rabbits [22], [23], hyperthyroidism promotes an up-regulation of SERCA 1 and a down-regulation of SERCA 2 in red muscle. An increase of Ca^2 +-ATPase content in hyperthyroid vastus lateralis muscle has also been detected in humans [39]. These measurements were performed using total muscle homogenates that do not allow the distinction between the two regions of the SR. We now show that the effect of hyperthyroidism varies depending on the SR fraction evaluated. With total homogenate we obtained the same results as those previously described (Figure 4A and B). However, in red muscle, the effect of hyperthyroidism in the two SERCA isoforms content was not the same in the light and heavy SR fractions. In the light fraction there was a ∼50% increase of SERCA 1 and no significant change of the SERCA 2 level (Figure 4 C and D). Thus, the variation of SERCA content in the light fraction does not match the pattern observed with the total homogenate. In the heavy SR fraction however, there was a 6 to 7 fold increase of SERCA 1 and a significant decrease of SERCA 2 (Figure 4 E and F). These data suggest that in hyperthyroidism the change of SERCA content in red muscle is observed mainly in the terminal cisternae and only a small modification is detected in the longitudinal part of the reticulum. In fact, the pattern observed in the heavy fraction is practically the same as that detected in the total homogenate (compare Figure 4A and B with 4E and F).

Figure 4.  Western blots showing SERCA isoform contents of red muscle from total homogenate, light SR fraction and heavy SR fraction. Immunodetection was obtained with SERCA 1 and SERCA 2 specific monoclonal antibodies. 20 µg of total homogenate (A and B), 10 µg of light SR vesicles (C and D) and 15 µg of heavy SR vesicles (E and F) were used to load the gels. The tissue homogenate were centrifuged at 10,000 g for 20 min and the supernatants were used. Hypo- and hyper- refers to samples obtained from hypo- or hyperthyroid animals. The densitometric values represent arbitrary units (A.U.) and the values represent the average ± SE. The number of Western blots performed varied between 3 and 8. The differences between control and either hyper- or hypothyroid were statistically significant (t-test) *p < 0.025, **p < 0.005 and ***p < 0.0005.

There was a reasonably good correlation between the variations of SERCA noted in Western blots and the Ca^2 + transport activity of the two red muscle fractions. In the light fraction, there was only a ∼50% increase of SERCA 1 and no variation in the level of SERCA 2. Accordingly, there was a ∼50% increase of both the rates of Ca^2 + uptake and Ca^2 +-dependent ATPase activity (Figure 5A and B, Table III). For the heavy fraction, the calcium transport rate depends on the balance between the two SERCA isoforms. In early reports it has been shown that vesicles containing SERCA 1 accumulate more Ca^2 + and at a faster rate than vesicles containing SERCA 2 [40], [41]. The Western blot analysis indicates a 6 fold increase of SERCA 1 and a 50% decrease of SERCA 2. As a result of these two divergent variations there was a 3-fold increase of both Ca^2 + transport and ATPase activity (Figure 5C and D, Table III).

Figure 5.  Ca^2 +-uptake and Ca^2 +-ATPase activity of light and heavy vesicles derived from red muscle. A and C refer to ⁴⁵Ca^2 +-uptake from light vesicles and heavy vesicles respectively. B and D refer to thapsigargin sensitive Ca^2 +-ATPase activity from light and heavy vesicles. The assay medium composition was described in Figure 3 and for Ca^2 +-ATPase activity we added trace amounts of ³²ATP. The vesicles used in the experiment were derived from Control (○), hypothyroid (Δ) and hyperthyroid (•) animals. The figure shows a typical experiment.

Table III.  Ca^2 +-uptake and Ca^2 +-ATPase activity from red skeletal muscle.

	Light Fraction		Heavy Fraction
Groups	Ca^2 +-uptake (µmolCa^2 +/mgprotein.min^−1)	Ca^2 +-ATPase (µmol Pi/mg protein.min^−1)	Ca^2 +-uptake (µmolCa^2 +/mg protein.min^−1)	Ca^2 +-ATPase (µmol Pi/mg protein.min^−1)
ConT	0.94±0.09 (26)	0.44±0.05 (22)	0.22±0.02 (14)	0.20±0.02 (13)
HypoT	0.32±0.09^a (10)	0.21±0.02^a (13)	0.26±0.03 (6)	0.18±0.02 (5)
HyperT	1.45±0.18^b (6)	0.67±0.08^b (9)	0.67±0.01^c (9)	0.58±0.06^c (10)

Assay medium and experimental conditions were as described in Figure 3. Values are mean±SE. The numbers of experiments are in parenthesis. ^aDifference between hypothyroid and both control and hyperthyroid, p<0.001; ^bDifference between hyperthyroid and control, p<0.01; ^cDifference between hyperthyroid and both, control and hypothyroid, p<0.001.

Rabbit white muscle expresses only SERCA 1 and the level of this protein did not vary significantly in either the total homogenate or in the two SR fractions (Figure 6). Similar to the Western blot, hyperthyroidism did not promote a significant change in the Ca^2 + uptake rate of the two SR fractions (Figure 7A and C). However, in both fractions, there was an increase of Ca^2 +-ATPase activity, which was not accompanied by a decrease of Ca^2 + transport, indicating that hyperthyroidism promotes an uncoupling of SERCA 1 (Figure 7B and D, Table IV). In a previous report [22] we did already observe that hyperthyroidism produces an increase in the SERCA 1 uncoupled ATPase activity in the light SR vesicle, but in our early work, the enhancement of ATPase activity promoted by hyperthyroidism was accompanied by a decrease of Ca^2 +-uptake, which was not detected in Table IV and Figure 7A. We do not know at present the reason for this discrepancy. One possibility is variability among the different groups of animals used.

Figure 6.  Western blots showing SERCA isoform contents of white muscle from total homogenate, Light SR Fraction and Heavy SR fraction. Immunodetection was obtained with SERCA 1 specific monoclonal antibodies. 8 µg of total homogenate (A), 0.5 µg of light SR vesicles (B) and 1 µg of heavy SR vesicles (C) were used to load the gel. The tissue homogenate were centrifuged at 10,000 g for 20 min and the supernatants were used. HypoT and HyperT refer to samples obtained from hypo- or hyperthyroid animals. Densitometric analysis showing arbitrary units (A.U.) relative to control. The figure shows the average±SE of 3 and 4 different experiments.

Figure 7.  Ca^2 +-uptake and ATPase activity of light and heavy vesicles derived from white muscle. A and C refer to ⁴⁵Ca^2 +-uptake from light vesicles and heavy vesicles respectively. B and D refer to thapsigargin sensitive Ca^2 +-ATPase activity from light and heavy vesicles. The assay medium composition was described in Figure 3 and for Ca^2 +-ATPase activity we added trace amounts of ³²ATP. The vesicles used in the experiment were derived from Control (○), hypothyroid (Δ) and hyperthyroid (•) animals. The figure shows a typical experiment.

Table IV.  Ca^2 +-uptake and Ca^2 +-ATPase activity from white skeletal muscle.

	Light Fraction		Heavy Fraction
Groups	Ca^2 +-uptake (µmolCa^2 +/mg protein.min^-1)	Ca^2 +-ATPase (µmol Pi/mg protein.min^-1)	Ca^2 +-uptake (µmolCa^2 +/mg protein.min^-1)	Ca^2 +-ATPase (µmol Pi/mg protein.min^-1)
ConT	4.67±0.42 (34)	1.75±0.09 (35)	2.02±0.13 (3)	1.67±0.13 (6)
HypoT	4.43±0.25 (22)	1.49±0.12 (14)	1.78±0.08 (3)	1.57±0.18 (6)
HyperT	4.34±0.33 (14)	2.24±0.18^a,b (14)	2.42±0.22^c (3)	2.04±0.14^d (6)

Assay medium and experimental conditions were as described in Figure 3. Values are mean±SE. The numbers of experiments are in parenthesis. ^aDifference between hyperthyroid and control, p<0.01; ^bDifference between hyperthyroid and hypothyroid, p<0.001; ^cDifference between hypothyroid and hyperthyroid, p<0.02. ^dDifference between hyperthyroid and control and hypothyroid p<0.05.

Hypothyroidism

The information available on hypothyroidism is scant and, as far as we know, the studies available on the effect of thyroid status on SERCA were performed only in rats. Hypothyroidism was shown to reduce Ca^2 +-ATPase in rat soleus muscle [42], and this decrement is mainly due to the decreased SERCA 1 expression [20]. We confirm these findings in rabbit red muscle (Figure 4 A and B) and in addition observed that the decrease of SERCA 1 is confined to the light fraction (Figure 4C and D). The levels of both SERCA 1 and SERCA 2 in the heavy fraction of hypothyroid rabbit red muscle were the same as those detected in control animals (Figure 4 E and F). Accordingly, Figure 5A and B and Table IV show that hypothyroidism promoted a decrease of both the rate of Ca^2 + uptake and Ca^2 +-dependent ATPase activity of the light SR vesicles but had no effect on Ca^2 + transport of the heavy SR fraction. In white muscle, hypothyroidism did not promote any measurable changes in SERCA 1 expression (Figure 6), Ca^2 + uptake or Ca^2 +-ATPase activity (Figure 7, Table IV).

Final remarks

In humans, thyroid disease promotes abnormalities in skeletal muscle function. The contraction and relaxation rates are increased in hyperthyroidism and decreased in hypothyroidism. The muscles more affected by thyroid hormone are those used for maintenance of posture, which are predominantly red muscles [42], [43]. A typical symptom is the shortening of the Achilles-tendon reflex time in hyperthyroidism and its prolongation in hypothyroidism [42]. These alterations correlate well with the changes of the SERCA noted in this study. We observed a higher SERCA 1 content in hyperthyroid red muscle, specifically in the heavy SR fraction, which is a portion of the reticulum involved in the muscle excitation-contraction coupling. Hudecova et al. [44] observed that thyroid hormone status promote alterations in the mRNA level of other proteins found in the heavy SR fraction. These include the ryanodine channel, the Na⁺/Ca^2 + exchanger and the IP3 channel. In conclusion, in this report we show that regulation of SERCA isoforms by thyroid hormones are not only muscle specific but also vary depending on the sub cellular compartment of the reticulum analyzed. These changes might correspond to one of the molecular basis of the altered contraction-relaxation rates detected in thyroid dysfunction.

This work was supported by grants from PRONEX-Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). APA and GMO are recipient of the CNPq fellowship. The authors are grateful to Prof. Paulo Arruda for helpful discussion and Mr V. A. Suzano and A.C. Miranda for technical assistance.