THYROIDVolume 11, Number 6, 2001Mary Ann Liebert, Inc.

Thyroid Hormone Stimulates Myoglobin Expression inSoleus and Extensorum Digitalis Longus Muscles of Rats:

Concomitant Alterations in the Activities of Krebs CycleOxidative Enzymes

Rosangela A. dos Santos, Gisele Giannocco, and Maria Tereza Nunes

Myoglobin (Mb) gene expression, Citrate Synthase (CS) and Succinate Dehydrogenase (SDH) activities of Soleus(S) and Extensorum Digitalis Longus (EDL) muscles were studied in intact, thyroidectomized and T3-treated(25ug/100g, BW, ip, 15 days) rats. The fiber type composition of S muscle was also evaluated and used as con-trol of the T3-induced effects. In the S muscle, the T3 treatment increased the Mb mRNA and protein expres-sion, as well as the CS and SDH activity. These changes occurred parallel to the expected increase in type II(fast) and decrease in type I (slow)-fibers in S muscle. In the hypothyroid state, the Mb mRNA was decreased,while the Mb expression and CS activity tended to decrease. In contrast the SDH activity was increased, prob-ably due to the enhanced motor activity that occurs as a short-term response to the hypothermia induced byhypothyroidism. In the EDL, the alterations were milder than those in S muscle in both thyroid states. Thesefindings show that Mb gene expression is induced by T3. This is concomitant with the enhancement of KrebsCycle enzyme activities and provides additional evidence that thyroid hormone increases the aerobic potentialof skeletal muscles, as well as the speed of muscle contraction.

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Introduction

SKELETAL MUSCLES can be characterized by their shorteningvelocity as being slow or fast. This is primarily determined

by the expression of specific myosin heavy chain (MHC) iso-forms in individual fibers, for example slow-type I and fast-type II MHC (1,2). In this regard, the muscle fiber types canbe classified in slow (type I) or fast-twitch (type II), which canbe easily differentiated on the basis of their histochemical re-action for the myosin-ATPase system, whose activity is alkali-labile in type I and alkali-stable in type II fibers (3,4).

Type I fibers are characterized further by having high ox-idative capacity, represented by higher mitochondrial en-zyme activities and increased expression of myoglobin (Mb)(5) that functions as a muscular store of oxygen (6,7) and fa-cilitates the diffusion of oxygen from the vascular system tomuscle mitochondria (8). On the other hand, fast-twitchfibers display high glycolytic enzyme activities and highglycogen content, which is consistent with their mechanicalproperties (5).

Thyroid hormones are known to influence the contractileand mechanical characteristics of skeletal muscle (4). In thesoleus (S) muscle, a typical slow-twitch (type I) muscle, tri-iodothyronine (T3) was shown to increase the activity and ex-pression of the alkali-stable myofibrillar adenosine triphos-phatase (ATPaSE) (fast-type II MHC) (4,9,10) and of thesarcoplasmic reticulum calcium transport systems (4,11,12).These proteins are markedly expressed in fast-twitch (type II)muscles such as extensorum digitalis longus (EDL).

The metabolic properties of skeletal muscles are correlatedto their functional role, therefore T3-induced alteration in theS muscle phenotype raises the question if its metabolic ox-idative properties remain constant or change due to anadapting mechanism to support the acquired new proper-ties.

Studies on the oxidative capacity of S muscle have beencarried out in thyroid hormone-treated rats but the resultsobtained are variable. Increased or unaltered succinate de-hydrogenase (SDH) activity (10,13) and unchanged or in-creased citrate synthase (CS) activity (14–16) have been re-ported, which might be due to differences in the measure-ment procedures used.

Considering that Mb is one of the principal cellular con-stituents that can influence the aerobic potential of muscle,this protein is useful as a good marker for the oxidative ca-pacity of this tissue. The present study was undertaken inan attempt to evaluate the effects of thyroid hormone on theMb mRNA and protein expression in S and EDL muscle ofrats. At the same time, thyroid hormone effects on the ac-tivity of the mitochondrial markers SDH and CS and on thefiber type composition were evaluated.

Here we report that T3 enhanced Mb gene expression, to-gether with increased activity of certain Krebs cycle en-zymes, providing additional evidence that thyroid hormoneincreases the aerobic potential of skeletal muscles. These ef-fects occur in spite of the increase in type II fibers, at the ex-pense of type I fibers, which determines the increased speedof muscle contraction.

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Materials and Methods

Animals and treatments

Male Wistar rats weighing 200 to 250 g were obtained fromour own breeding colony and maintained on rat chow andtap water ad libitum. They were housed in a room kept at con-stant temperature (23 6 1°C) and on a 12-hour-light/12-hourdark (lights on at 7:00 A.M.) schedule. The animals were madehypothyroid by surgical thyroidectomy (Tx), after beingdeeply anesthetized with ketamine (15 mg/kg body weight,intraperitoneally) and xylazine (3 mg/kg body weight, in-traperitoneally) and received 0.03% methylmercaptoimida-zole (MMI; Sigma Chemical Co., St. Louis, MO) and 0.5% cal-cium chloride (CaCl2; Sigma) in the drinking water for 15 days.Euthyroid sham-operated animals were used as a controlgroup. Experimental hyperthyroidism was induced in sham-operated rats by intraperitoneal administration of T3 (Sigma)at a dose of 25 mg per 100 g of body weight for 15 days. Eu-thyroid and hypothyroid animals received intraperitoneal ad-ministration of vehicle (saline). Rats (five per group) wereanesthetized and killed by decapitation after approximately15 hours of the last T3 or saline injection. Blood samples werecollected to evaluate the concentrations of total T3 by ra-dioimmunoassay (RIA) (RIA-gnost T3 kit, CIS Bio Interna-tional, Gif-Sur-Yvette Cedex, France) in the sera. The hindlimbS and EDL were used as slow-twitch/oxidative and fast-twitch/glycolytic muscles, respectively. After decapitation,the S and EDL muscles were quickly excised, freed from con-nective tissue and fat, and prepared for the specific studies.

The experimental protocol conforms with ethical princi-ples in animal research adopted by the Brazilian College ofAnimal Experimentation (COBEA) and was approved by theBiomedical Sciences Institute/University of São Paulo-Ethi-cal Committee for Animal Research (CEEA).

Procedures

Evaluation of Mb expression levels. The Mb expression inS and EDL muscles was studied by Northern and Westernblot analysis.

Northern blot analysis.Total RNA was isolated using the acidguanidinium thiocyanate-phenol-chloroform extractionmethod and quantified by absorbance at 260 nm (17). TotalRNA samples were denatured with formaldehyde-for-mamide, electrophoresed in 1% agarose gels containing 2.2M formaldehyde in 13 3-N-morpholino-propanesulfonicacid buffer and blotted to a nylon membrane (Nylon-1 mem-brane, Gibco BRL, Rockville, MD) by neutral capillary trans-fer. The membrane was baked at 80°C for 2 hours in a vacuum oven and prehybridized in 50% formamide hy-bridization solution and 100 mg/mL denatured salmon spermDNA at 42°C for 4 hours (18). Subsequently the membranewas probed with a 32P-labeled rat Mb cDNA (GenBankAF197916; online: http://www.ncbi.nlm.nih.gov/ [Novem-ber 3 1999]) by random priming (Random Primers DNA La-beling System Kit, Gibco BRL) for 16 hours at 42°C. The mem-brane was washed under high stringency conditions andsubjected to autoradiography and quantified by phospho-rimaging, using ImageQuant software (Molecular Dynamics,Sunnyvale, CA). All blots were stripped and rehybridizedwith a 32P-labeled RNA probe specific for 18S ribosomal RNA(18S c-rRNA), synthesized by in vitro transcription (Maxi

Script In vitro transcription kit, Ambion, Austin, TX), to cor-rect for the variability in RNA loading. The results were ex-pressed as mean 6 SEM of Mb mRNA/18S c-rRNA ratio.

Western blot analysis. The S and EDL muscles were homo-geneized in an Omni-mixer (Omni International, Warrenton,VA) in the appropriate buffer (0.3 M Sucrose; 0.1 M KCl; 20mM Tris-HCl, pH 7.0). The homogenate was centrifuged for60 minutes at 100,000g (Ti 80 Beckman rotor) and the pro-tein content of the supernatant (cytosolic fraction) was de-termined (19). Cytosolic protein samples (30 mg per lane)were treated with Laemmli sample buffer (20), heated in aboiling water bath for 4 minutes and resolved by sodium do-decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 5% acrylamide stacking gel and a 15% acry-lamide resolving gel. Electrotransfer of proteins from the gelto nitrocellulose (Hybond-C, Amershampharmacia Biotech,Little Chalfont) was performed for 90 minutes at 300 mA.Nonspecific protein binding to the nitrocellulose was re-duced by preincubating the filter 2 hours at room tempera-ture in blocking buffer (1% nonfat dry milk, 137 mM NaCl,

SANTOS ET AL.546

FIG. 1. Effect of thyroid hormone on mRNA and Proteinlevels of myoglobin detected by Northern blot and Westernblot, respectively. A: autoradiographs of representativeNorthern blot analysis using (32P)CTP-labeled rat myoglo-bin cDNA and (32P)UTP-labeled 18S c-rRNA as probes (leftpanel) and Western blot of protein extracts hybridized witha human anti-Mb antibody (right panel). Each lane wasloaded with 10 ug of total RNA (Northern blot) or 30 ug oftotal protein (Western blot) from soleus (S) muscles of (1)Hypo (thyroidectomy 1 0.03% metimazole), eu (sham-oper-ated) and hyperthyroid (25 ug T3/100g BW, ip, 15 days) an-imals. B: summary data of densitometric estimates of MbmRNA normalized with 18S rRNA or densitometric analy-sis of Mb protein bands from Western blot. The data shownare the average 6 SEM. *p , 0.01 compared with euthyroidgroup (ANOVA, plus Student Newman-Keuls test).

B

A

2.7 mM KCl, 8 mM NaHPO4, 1.4 mM KH2PO4, and 0.02%Tween 20). The nitrocellulose blot was incubated with hu-man antimyoglobin antiserum (1/1000; Sigma), diluted inblocking buffer overnight at 4°C and washed for 60 minuteswith the blocking buffer without milk. The blots were thenincubated with 2 mCi [125I] Protein A (30 mCi/mg) (Amer-sham-Pharmacia) in blocking buffer for 3 hours at room tem-perature and then washed again for 2 hours as describedabove. [125I] Protein A bound to the antimyoglobin antibodywas detected by storage phosphor autoradiography andquantified, in arbitrary units, using a laser-scanning den-sitometer (Molecular Dynamics, Sunnyvale, CA).

Evaluation of the CS and SDH activities. The CS activity(CS, E.C. 4.1.3.7) was determined as previously described(21,22). The extraction buffer for CS contained 50 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and0.05% (v/v) Triton X-100; pH was 7.4. The assay buffer ofCS consisted of 100 mM Tris-HCl, 0.2 mM 5,59-dithio-bis-2-nitrobenzoic acid, 15 mM acetyl-CoA, and 0.5 mM oxaloac-

etate, pH 8.1. The final volume of the assay mixture was 1mL. CS was assayed by following the rate of change in ab-sorbance at 412 nm. The spectrophotometric measurementswere performed using a Gilford recording spectrophotome-ter (Response model) at 25°C. Preliminary experiments es-tablished that extraction and assay procedures producedmaximum enzyme activity for the enzyme studied. The en-zyme activity was expressed as nanomoles of substrate uti-lized per milligram of total protein per minute.

The SDH activity (SDH, E.C. 1.3.99.1) of the S and EDL mus-cles was determined by histochemistry as described (22,23).In brief, the muscles were removed and immediately frozenin melting isopentane and stored at 280°C. Frozen muscleswere cut through the medial region using a cryostat (10-mmcross-sections). Alternate serial sections were subjected to re-actions for SDH (23). The enzyme activities were measured insingle fibers of S and EDL muscles by quantifying the for-mazan reaction product by means of the program Image 1.55,NIH, processed in a MacIntosh Quadra 950 computer. At least1,500 fibers were counted per muscle, in a total of 5 musclesper group, by two different observers. Based on the degree ofSDH expression, the fibers were classified as being SDH2 (ab-sent), SDH1 (moderate) or SDH11 (high).

Evaluation of the effectiveness of the treatments. The ef-fectiveness of the treatments in the different experimentalgroups was evaluated by determining the T3 serum levelsby RIA, using a standard curve prepared adding differentconcentrations of T3 in iodothyronines-free rat serum, andby histochemical analysis (3,24) of the fiber type composi-tion of the medial region of soleus muscle, in which thyroidhormones are known to increase expression of major histo-compatibility complex (MHC) type II and to decrease that oftype I (4,10). At least 1,500 fibers were counted per muscle,in a total of 5 muscles per group, by two different observers.

Statistics

The data concerned to the CS activity and serum concen-trations of thyroid hormone were subjected to analysis ofvariance (ANOVA), followed by the Student-Newman-Keuls test. For the analysis of the SDH and myofibrillar ATPase activities, the data were subjected to analysis of fre-

T3 EFFECTS ON MYOGLOBIN AND MITOCHONDRIAL ENZYMES 547

B

A

FIG. 3. Maximal activities of citrate synthase in soleus (S)and extensorum digitalis longus (EDL), of hypothyroid, eu-thyroid, and hyperthyroid (25 mg triiodothyronine (T3)/100g body weight, intraperitoneally, 15 days) rats. The valuesare expressed as nanomoles per minute per milligram of to-tal protein. The data shown are the average 6 SEM. *p ,0.01 compared with euthyroid group (ANOVA, plus StudentNewman-Keuls test).

FIG. 2. Effect of thyroid hormone on mRNA and Protein lev-els of myoglobin detected by Northern blot and Western blot,respectively. A: autoradiographs of representative Northernblot analysis using (32P)CTP-labeled rat myoglobin cDNA and(32P)UTP-labeled 18S c-rRNA as probes (left panel) and West-ern blot of protein extracts hybridized with a human anti-Mbantibody (right panel). Each lane was loaded with 10 ug of to-tal RNA (Northern blot) or 30 ug of total protein (Westernblot) from extensorum digitalis longus (EDL) muscles of (1)Hypo (thyroidectomy 1 0.03% metimazole), eu (sham-oper-ated) and hyperthyroid (25 ug T3/100g BW, ip, 15 days) ani-mals. B: summary data of densitometric estimates of MbmRNA normalized with 18S rRNA or densitometric analysisof Mb protein bands from Western blot. The data shown arethe average 6 SEM. *p , 0.01 compared with euthyroid group(ANOVA, plus Student Newman-Keuls test).

quences by x2 test. Data are expressed as means 6 SEM. Dif-ferences were considered significant at p , 0.05.

Results

The effectiveness of thyroidectomy and T3 treatment waschecked by determining the serum T3 concentration, whichwas at least one-third lower (0.10 6 0.01 ng/mL; n 5 5; p ,0.01) and 6-fold higher (2.35 6 0.94 ng/mL; n 5 5; p , 0.01)in the hypothyroid and hyperthyroid animals, respectively,than those of the control group (0.37 6 0.08 ng/mL; n 5 5),and also by the increase in the percentage of fast type II MHCfibers (A and X) from 16.08 6 0.89 to 28.63 6 2.78% (p , 0.05)and decrease in the percentage of slow-type I MHC fibersfrom 84.59 6 1.14 to 71.37 6 1.66% (p , 0.05) in the S mus-cle of T3-treated rats. These data are in accordance with pre-vious studies in which the well known effects of T3 on mus-cle fiber type composition were described (4,9,10) andconfirm the induction of hypothyroidism and hyperthy-roidism in our experimental groups.

Effect of thyroid state on Mb expression

The effect of thyroid hormone on Mb mRNA and proteinexpression in S muscle is shown in Figure 1. The Mb mRNAcontent increased at least 50% in T3-treated rats, while a re-duction around 75% was observed in hypothyroidism. TheMb content of the S muscle paralleled the mRNA expressionin T3-treated and hypothyroid rats, although in the thyroid-deficient state the decrease in the Mb content was at the limitof significance.

As shown in Figure 2, in EDL muscle of the T3-treated ratsno alteration in Mb mRNA expression was observed; nev-ertheless, an increase in the Mb content was detected. In thehypothyroid state, the Mb mRNA abundance was shown tobe decreased, even though no reduction was detected in theamount of the protein. These data indicate that the basal MbmRNA expression depends on physiological levels of thy-roid hormone and that T3 induces an increase in the Mb con-tent in S and EDL muscle, although the effect is more pro-nounced in S.

Effect of thyroid state on mitochondrial enzyme activities

The CS activity in S and EDL muscles of rats subjected tohyperthyroidism or hypothyroidism is presented in Figure3. T3 treatment induced a significant increase in CS activityof both muscles. In the hypothyroid state, the CS activity wasdecreased in the EDL, and tended to decrease in S muscle.

Table 1 shows the SDH activity of S muscle in rats sub-jected to T3 treatment and hypothyroidism. It was observedthat T3 treatment induced an increase in the number of fibersthat express SDH1 activity, in parallel to a decrease (46%) inthe number of SDH2 fibers. The number of fibers express-ing high SDH activity (SDH11) was unchanged. In hy-pothyroid rats, the number of fibers that express SDH1 ac-tivity was shown to be decreased, in contrast to the SDH11

fibers that were increased in number. The number of SDH2

fibers remained similar to the control group.The effect of thyroid hormone on the SDH activity of EDL

muscles is shown in Table 2. T3 treatment induced a decreaseof SDH1 fibers and increase in the SDH11 fibers, with nomodifications in the number of SDH2 fibers. In the hy-pothyroid state, the SDH1 fibers were decreased in number,whereas no significant modifications were detected in theSDH2 and SDH11 fibers.

Discussion

This study investigated, for the first time, the role of thy-roid hormone on the Mb mRNA and protein expression inrat S and EDL muscles and the possible physiological con-sequences of these effects. We have shown here that Mb geneexpression is under the control of thyroid hormone in slow-twitch muscle, as indicated by the decrease in the Mb mRNAand protein content observed in thyroidectomized rats andincrease of both parameters in T3-treated rats. The increasein Mb mRNA content with a corresponding elevated levelof Mb might suggest pretranslational regulation of Mb ex-pression rather than a reduced turnover of Mb mRNA sinceMb protein expression is enhanced.

The effect was less pronounced in the EDL muscle; we ob-served (1) decreased Mb mRNA content that was not followed

SANTOS ET AL.548

TABLE 1. SUCCINATE DEHYDROGENASE EXPRESSION (SDH) IN S MUSCLE OF HYPOTHYROID, EUTHYROID, OR HYPERTHYROID ANIMALS TREATED FOR FIFTEEN DAYS

Group SDH2 (%) SDH1 (%) SDH11 (%)

Hypothyroid 6.86 6 1.17* 65.17 6 2.80** 27.84 6 1.64**Euthyroid 6.80 6 1.34* 75.94 6 2.89** 17.26 6 2.74**Hyperthyroid 3.36 6 0.10* 79.65 6 0.98** 17.00 6 0.91**

Values are means 6 SEM.*p, 0.05; **p, 0.01.

TABLE 2. SUCCINATE DEHYDROGENASE EXPRESSION (SDH) IN EDL MUSCLE OF HYPOTHYROID, EUTHYROID, OR HYPERTHYROID ANIMALS TREATED FOR FIFTEEN DAYS

Group SDH2 (%) SDH1 (%) SDH11 (%)

Hypothyroid 51.04 6 1.14 21.15 6 0.71** 27.81 6 1.85*Euthyroid 46.72 6 0.68 29.13 6 0.65** 24.15 6 0.93*Hyperthyroid 44.85 6 1.60 24.71 6 0.66** 30.44 6 1.21*

Values are means 6 SEM.*p, 0.05; **p, 0.01.

by a decrease in the Mb content in thyroidectomized rats and(2) slight decrease in the Mb mRNA expression, followed byincrease in the protein amount, in T3-treated rats. This differ-ence in the T3 response between S and EDL muscles suggeststhat slow-twitch muscle is more sensitive to T3 than fast-twitchmuscle, which could be due to differences in the thyroid hor-mone receptors expressed in these muscles, as previously re-ported (9,25). Furthermore, the increase in Mb content with-out significant changes in mRNA expression could suggestthat, in EDL, the efficiency of mRNA translation to protein isincreased by T3 treatment. In fact, posttranscriptional effect ofT3 has been reported in some T3 target genes, such as the malicenzyme and growth hormone genes (26,27).

Considering that Mb may serve a variety of functions inmuscular oxygen supply, for example oxygen storage, facil-itated oxygen diffusion and myoglobin-mediated oxidativephosphorylation (6,7), its increased content in the hyperthy-roid state suggests that the amount and availability of oxy-gen to the metabolic reactions involving adenosine triphos-phate (ATP) synthesis in both slow- and fast-twitch muscleare increased. This could represent a functional benefit to theskeletal muscle since in this condition, ATPases includingthe sarcoplasmic reticulum calcium-ATPase (SERCA),Na1/K1-ATPase and the MHC II (28) are known to havetheir expression and activity increased by T3. In addition, thehigh Mb content in S and EDL muscles of T3-treated rats in-dicates that these muscles could be more resistant to fatigue.

Thyroid hormone actions are initiated by T3 binding to nu-clear receptors that bind to particular regions of the DNA,known as thyroid response elements (TREs), which have beenidentified in several genes, such as SERCA and the MHC multi-gene family, as already reported. Whether thyroid hormonesexert a direct effect on Mb gene expression or if the increasein Mb content is an adapting response to thyroid hormone ef-fects on the muscle metabolism deserves further investigation.

On the other hand, thyroid hormone is known to activategenes, whose transcription products could regulate the Mbgene expression. For instance, T3 positively regulates myo-genin gene expression (29) whose product, the myogenin, in-duces the myocyte-specific enhancer binding factor MEF-2(30), a transcriptional factor that acts synergistically with Sp1and binds to the Mb promoter, activating gene transcriptionduring muscle development and differentiation (31). Actu-ally, this should be considered in our study, since thyroidhormone effects on the adult skeletal muscle plasticity arevery well known (4,9,10,16).

The study of the Krebs cycle mitochondrial enzymes re-vealed that T3 treatment induced a significant increase in theCS activity in S and EDL muscles, although the fast-twitchmuscle response to thyroid hormone was lower than slow-twitch ones. Our results, with regard to the S muscle, are inaccordance with those from Winder and Holloszy (32),Ianuzzo et al. (33), Locke et al. (15) and McAllister et al. (34),which have shown an increase in CS activity (at least 1.5-fold) after T3 treatment compared to euthyroid rats.

We have also observed increased CS activity in EDL mus-cle of T3-treated rats, as well as Winder and Holloszy (32)and McAllister et al. (34), who have found the same alter-ation in other types of fast-twitch muscle (white section ofquadriceps and vastus lateralis, respectively); in contrast,Ianuzzo et al. (33) and Locke et al. (15) could not find anyalteration in the CS activity in the plantaris muscle, which isalso classified as a fast-twitch muscle. Although all those

cited muscles are classified as fast-twitch muscles, their pro-portion of fiber type composition differ, which could possi-bly explain the different responses to T3 besides the differ-ent doses and period of hormonal treatments.

CS is considered to be a flux-generating enzyme in the car-boxylic acid cycle (35) and is used together with cytochromeoxidase as a conventional marker of oxidative capacity. Its in-creased activity in S and, in a lesser degree, in EDL muscle ofthyroid hormone-treated rats, is additional evidence that thy-roid hormone increases the skeletal muscle oxidative capacity.

A stimulating effect of thyroid hormone administration onthe SDH activity of S and EDL muscles was also observed inour study. However, in hypothyroidism we observed a de-creased number of SDH1 fibers, concomitant with an unex-pected increase in the number of SDH11 fibers in S muscle.Taking into account that in thyroid hormone deficient states,heat production is impaired (36) and that the thermogenic ca-pacity of the brown adipose tissue (BAT) is limited (37), it isconceivable that an increase in the motor activity (shivering)might be achieved as an adapting physical short-term re-sponse to the hypothermia (38). In fact, there is evidence thatmotor activity is the key factor regulating SDH expression(39). Indeed, we observed a decrease in SDH11 activity(27.84 6 1.64 vs. 20.926 0.54%) lengthening the period of hy-pothyroidism to 30 days, which supports this assumption.

Anyway, the increased SDH11 activity in S muscle of an-imals that were hypothyroid for 15 days is likely to have norelevant functional significance, because we observed con-comitantly a lower Mb expression, which implies that thetransport and availability of oxygen for oxidative phospho-rylation by SDH are reduced.

In conclusion, we have shown that Mb gene expression isunder the control of thyroid hormone in S and, to a lesser de-gree, EDL muscles. Associated with the observed increasedCS and SDH activity, this provides additional evidence thatthyroid hormone increases the oxidative capacity of skeletalmuscles. Despite the new findings reported here more re-search is necessary to establish the physiological context ofthese adaptations in terms of thyroid-responsive gene regu-lation. Furthermore, the physiological relevance of the appar-ent controversial effect of T3 increasing the aerobic potentialin parallel with the velocity of shortening twitch contractionand relaxation of skeletal muscle deserves to be examined.

Acknowlegments

The authors are grateful to Dr. Alec Jeffreys, University ofLeicester, UK, for kindly providing the human Mb cDNAthat was used in our first experiments and to Dr. Bernd Glossfor helping us reviewing the manuscript.

The work was supported by a grant from the Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP92/0612-8). Rosangela A. Santos is a recipient of a fellowshipfrom the same foundation (FAPESP 96/12058-6; 97/8605-4).

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Address reprint requests to:Maria Tereza Nunes, Ph.D.

Department of Physiology and BiophysicsInstitute of Biomedical SciencesAv. Prof. Lineu Prestes, 1524

University of São Paulo05508-900, São Paulo

Brazil

E-mail: mtnunes@fisio.icb.usp.br