Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb
suspension
GARY M. DIFFEE, FADIA HADDAD, ROBERT E. HERRICK, AND KENNETH M. BALDWIN Department of Physiology and Biophysics, University of California at Irvine, Irvine, California 92717
DIFFEE, GARY M., FADIA HADDAD, ROBERT E. HERRICK, AND KENNETH M. BALDWIN. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension. Am. J. Physiol. 261 (Cell Physiol. 30): C1099-C1106, 1991.-The aim of this study was to contrast competing influences, hypothyroidism and hindlimb suspension, on myosin heavy chain (MHC) expression studied at the protein level and mRNA level. Female Sprague-Dawley rats were assigned to either normal control (NC), normal suspended (NS), or hypothyroid (thyroidectomized) control (TC) and suspended (TS) groups. NS and TS animals were suspended for 14 days following which myofibrils and total RNA were purified from the hindlimb muscles. In the soleus and vastus intermedius (VI), there was an increase in type I MHC and a decrease in type IIa MHC in both the TC and TS groups and a decrease in type I and increase in type IIa MHC in the NS group. At the mRNA level, similar shifts were observed with the exception that 1) the increased type IIa MHC seen in the soleus and VI of the NS animals was not accompanied by an increase in IIa mRNA and 2) type IIb mRNA was increased in the NS soleus without concomitant changes in IIb protein levels. These data suggest the following: 1) a hypothyroid state predominates over mechanical unweighting factors in the control of MHC distribution in slow muscles; and 2) translational or posttranslational factors may be important in the regulation of type IIa and IIb MHC expression during hindlimb suspension. skeletal muscle; myosin gene expression
isoforms;
myosin
heavy chain mRNA;
PROTEIN MYOSIN is a hexameric molecule consisting of two heavy chains and two pairs of light chains. Myosin exists as multiple isoforms as a result of polymorphic expression of both the myosin heavy chain (MHC) and myosin light chain (MLC) subunits. In mammalian striated muscles, the heavy chains are encoded by a highly conserved multigene family (23). At least six MHC genes are known to be expressed in rodent striated muscle, including the three heavy chains identified in adult rat skeletal muscle (type I, type IIa, and type IIb; Ref. 20). These isoforms are distributed throughout the musculature in a characteristic fashion, implying a functional role for the different isoforms (IO, 31). Indeed, the shortening velocity of single skeletal muscle fibers has been shown to be related to both the MHC (24,26) and MLC (12,26) composition of the fiber. Type I MHC has been generally associated with slowtwitch fibers and is thought to have a slower adenosinetriphosphatase (ATPase) activity, whereas the type IIa
THE CONTRACTILE
0363-6143/91
$1.50 Copyright
and IIb MHCs are found in faster contracting fibers. The phenotypic expression of MHC isoforms within a given muscle appears to be under the control of a number of factors; among them developmental (4, 18, 20), neural (5, 25), hormonal (9, 17, 20), and mechanical factors (3, 8, 30). However, little is known regarding the possible interaction between these various regulators of MHC content, or which, if any, predominate over others in the control of MHC expression. Information regarding the degree of cooperativity or competition between those forces that influence MHC isoform distribution promises to greatly enhance our understanding of the overall control of MHC gene expression. Our interest in this study was to contrast two competing influences that have been well characterized with regard to their impact on myosin isoform expression: hypothyroidism and hindlimb suspension. The hormone thyroxine has been shown to be a potent, tissue-specific regulator of MHC gene expression (14,16, 17, 20). Izumo et al. (17) demonstrated that, in slow muscle such as the soleus, hyperthyroidism was associated with an upregulation of the expression of the type IIa MHC messenger RNA, whereas hypothyroidism was associated with an upregulation of type I MHC expression. In fast muscles, hypothyroidism was associated with an increase in type IIa MHC mRNA. These responses to altered thyroid state have also been characterized at the protein level, as Fitzsimons et al. (9) demonstrated that, in slow muscles, hyperthyroidism was associated with an increase in the relative amount of fast myosin, whereas hypothyroidism resulted in a relative increase in the slow myosin content. In addition, slow muscle was found to be much more responsive to alterations in thyroid state than was fast muscle (9). Mechanical factors, such as the load imposed on a muscle, are also thought to have their greatest impact on slow muscles. Removal of weight-bearing activity from slow muscles, as in the hindlimb suspension model (8, 29) or during exposure to a zero gravity environment (3), results in a shift in isomyosin distribution characterized by an increase in the relative amount of the intermediate and the fast myosins (containing the type IIa and IIb MHCs). Conversely, increasing the degree of weight bearing activity through surgical removal of the synergists has been shown to increase the relative amount of slow myosin (containing type I MHC) significantly (30). However, while the effects of hindlimb suspension on myosin isoform distribution have been well character-
0 1991 the American
Physiological
Society
Cl099
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.151.010.035) on October 22, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
Cl100
HYPOTHYROIDISM
AND
SUSPENSION
ized, relatively little is known regarding the impact of suspension on MHC distribution at the mRNA level. Thomason et al. (28) reported that, although type I MHC protein levels had been shown to be decreased following 7 days of suspension, type I MHC mRNA levels did not fall, thus raising the possibility of translational or posttranslational regulation of MHC expression during the course of hindlimb suspension. Given the above, our objectives in this study were to I) determine the relative efficacy of hypothyroidism and hindlimb suspension in the modulation of MHC expression when these two manipulations are imposed simultaneously in an animal and 2) compare the response with these manipulations at both the mRNA level and at the protein level to distinguish between transcriptional and translational control of MHC expression. Here we present evidence that thyroid state predominates over mechanical factors associated with unweighting in the determination of MHC isoform expression. Evidence is also presented suggesting incon gruities in the response to hindlimb suspension of the MHC isoforms vs. their respective mRNA levels. METHODS
Animal care and suspension. Eighteen normal and eighteen thyroidectomized adult female Sprague-Dawley rats (-250 g) were obtained from Taconic Farms (Germantown, NY). Thyroidectomized animals were immediately placed on a regimen of intraperitoneal injections of the antithyroid drug 6-n-propyl-2-thiouracil (PTU, 12 mg/kg body wt) every other day. Animals were housed in temperatureand light-controlled quarters and received food and water ad libitum. All experiments and procedures were approved by the University of California-Irvine Animal Use Committee and conformed to National Institutes of Health guidelines for the humane treatment of animals. Approximately two weeks following the thyroidectomy, the resting metabolic status of the animals was tested to verify an altered thyroid state. Resting oxygen uptake (VO,) was measured according to methods described previously (22). After the determination of metabolic status, animals were assigned to one of four groups: normal control (NC), hypothyroid control (TC), normal suspended (NS), and hypothyroid suspended (TS). Animals in the NS and TS groups were suspended by an .oninvasive procedure that has been described previously (29). Briefly, a tail cast was applied to the animal, leaving the distal one-third of the tail free to allow for proper thermoregulation. The tail cast was attached to a hook at the top of the cage, and the height of the hook was adjusted so that only the front legs could contact the cage floor. This method allows the animals full 360” rotation as well as access to food and water without allowing the hindlimbs to contact the cage floor or walls. Suspension was discontinued and the animal removed from the study if any discoloration of the tail was detected. At the end of 2 wk of hindlimb suspension, the animals were anesthetized with pentobarbital sodium and blood was drawn for plasma 3,5,3’-triiodothyronine (T3) analy-
EFFECTS
IN
SKELETAL
MUSCLE
sis. The animals were then killed with a lethal overdose of pentobarbital sodium, and the heart and the soleus, plantaris, and vastus intermedius (VI) muscles were removed. The heart and the muscles from one leg of each animal were trimmed of connective tissue, weighed, placed into precooled glycerol, and stored at -20°C for subsequent myofibril purification, while the muscles of the other leg were quick frozen and stored at -70°C for RNA extraction. Myofibril isolation and gel electrophoresis. Myofibril purification and protein determination were performed according to a method described previously (27,31). After purification, myofibrils were stored at a protein concentration of 1 mg/ml in storage buffer consisting of 50% glycerol, 100 mM Na4P207, 5 mM EDTA, and 2 mM 2mercaptoethanol (pH 8.8). MHCs were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to a modification of the method described by Danielli-Betto et al. (7). Briefly, the protein sample was mixed with sample buffer that consisted of 62.5 mM tris(hydroxymethyl)aminomethane (Tris), 20% glycerol, 1% 2-mercaptoethanol, 2.3% SDS, and 0.05% bromophenol blue. This mixture was heated at 100°C for 2 min and then -1 pg of protein was loaded onto the gel. The stacking gel was 4% total acrylamide, 2.6% bis-acrylamide (expressed as a percent of the total acrylamide), and 40% glycerol, pH 6.8, whereas the resolving gel was 7% acrylamide, 2.6% bis-acrylamide, and 40% glycerol, pH 8.3. The gels were run at 120 V (constant voltage) for 4-4.5 h until the dye front migrated off of the gel. To determine the cardiac native myosin isoform distribution, the myofibril fraction isolated from the heart was subjected to nondissociating PAGE for 20 h according to the method described previously (27, 31). Both SDS gels and native gels were stained for 1 h in 0.1% Coomassie Blue R-250, 25% methanol, and 8% acetic acid. Destaining occurred by diffusion until the background was clear. After staining and destaining, the gels were scanned densitometrically at 630 nm in a Zeineh soft laser densitometer (BioMedical Instruments, Fullerton, CA) interfaced to an IBM PC computer equipped with software to perform peak area integration analysis. The areas of the peaks corresponding to each band were summated and then the area for each peak expressed as a percent of the total area. In this way, each native myosin or MHC isoform is expressed as a percent of the total myosin or total MHC present on the gel. RNA isolation. Total cellular RNA was isolated from the muscle samples using the guanidium isothiocyanatecesium chloride, ethanol precipitation method (6, 11). The RNA pellet was suspended in diethyl pyrocarbonate pretreated water and stored frozen at -20°C for slot blot analysis. RNA quantities were determined by ultraviolet (UV) absorption at 260 nm. RNA quality was routinely checked by electrophoresis of 0.5-1.0 pugof RNA through a 1% agarose gel in lx Tris-borate-EDTA buffer containing ethidium bromide. The gels were exposed to UV light and checked for the presence of two fluorescing bands, corresponding to the 28s and 18s ribosomal RNA. No evidence of RNA degradation was observed in any of
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.151.010.035) on October 22, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
HYPOTHYROIDISM
AND
SUSPENSION
the samples tested. Preparation of synthetic probes. MHC gene specific oligonucleotides, 20 bases in length, were purchased from the University of California-Irvine Biological Chemistry Department. The sequences of these probes have been reported previously (14). These sequences are located in the 3’-untranslated regions of the MHC isoform mRNAs and have been shown to be highly isoform specific (14). The probes were Y-end labeled with 32P to a specific activity of 4-5 x lo6 cpm/mol by the T4-polynucleotide kinase reaction (21). Labeled oligonucleotides was separated from unlabeled and from unincorporated [T-~~P]ATP by urea PAGE. The probes were repeatedly tested for specificity using Northern blot analysis with both . . 1 . . n * * positive and negative controls for each probe. RNA slot-blot assay. Total RNA (l-2 pg) was blotted onto a Nytran nylon membrane (Schleicher & Schuell, Keene, NH) using a standard slot-blot procedure. The filters were subsequently fixed with UV irradiation. Prehybridization, hybridization, and washing procedures for the blots were all carried out according to the manufacturers recommendations. After the final wash, the membranes were autoradiographed with an intensifying screen at -70°C for l-4 days. mRNA levels were quantified by densitometric scanning of the autoradiograms. The quantified amount of each MHC mRNA in each of the experimental groups was then normalized to the level found in the NC group so that the amount of each MHC mRNA was expressed as a percent change from normal control levels. Plasma T3 determination. Plasma T3 levels in blood samples drawn just prior to death were determined using a commercial radioimmunoassay kit (Gamma Coat [1251] T3, Dade Diagnostics, Cambridge, MA). Statistical analysis. All data are presented as means t SE from several muscles. Data were analyzed using a one-way analysis of variance to determine homogeneity of variance. For each variable measured, data from the experimental groups were tested for significant difference compared with the NC group data. In some instances, tests of significance were also made between data from the TC group and data from the TS group. P values of ~0.05 were considered significant after adjustment using the Bonferroni correction for multiple comparisons (32).
EFFECTS
IN
SKELETAL
Cl101
MUSCLE
1. Evidence for existence of hypothyroid state
TABLE
NC
n Body weight, g Plasma TB, ng/dl Heart weight, mg Cardiac isomyosins, relative % VI
NS
TS
9
9
9
9
299t4
246t4*
suspension
GARY M. DIFFEE, FADIA HADDAD, ROBERT E. HERRICK, AND KENNETH M. BALDWIN Department of Physiology and Biophysics, University of California at Irvine, Irvine, California 92717
DIFFEE, GARY M., FADIA HADDAD, ROBERT E. HERRICK, AND KENNETH M. BALDWIN. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension. Am. J. Physiol. 261 (Cell Physiol. 30): C1099-C1106, 1991.-The aim of this study was to contrast competing influences, hypothyroidism and hindlimb suspension, on myosin heavy chain (MHC) expression studied at the protein level and mRNA level. Female Sprague-Dawley rats were assigned to either normal control (NC), normal suspended (NS), or hypothyroid (thyroidectomized) control (TC) and suspended (TS) groups. NS and TS animals were suspended for 14 days following which myofibrils and total RNA were purified from the hindlimb muscles. In the soleus and vastus intermedius (VI), there was an increase in type I MHC and a decrease in type IIa MHC in both the TC and TS groups and a decrease in type I and increase in type IIa MHC in the NS group. At the mRNA level, similar shifts were observed with the exception that 1) the increased type IIa MHC seen in the soleus and VI of the NS animals was not accompanied by an increase in IIa mRNA and 2) type IIb mRNA was increased in the NS soleus without concomitant changes in IIb protein levels. These data suggest the following: 1) a hypothyroid state predominates over mechanical unweighting factors in the control of MHC distribution in slow muscles; and 2) translational or posttranslational factors may be important in the regulation of type IIa and IIb MHC expression during hindlimb suspension. skeletal muscle; myosin gene expression
isoforms;
myosin
heavy chain mRNA;
PROTEIN MYOSIN is a hexameric molecule consisting of two heavy chains and two pairs of light chains. Myosin exists as multiple isoforms as a result of polymorphic expression of both the myosin heavy chain (MHC) and myosin light chain (MLC) subunits. In mammalian striated muscles, the heavy chains are encoded by a highly conserved multigene family (23). At least six MHC genes are known to be expressed in rodent striated muscle, including the three heavy chains identified in adult rat skeletal muscle (type I, type IIa, and type IIb; Ref. 20). These isoforms are distributed throughout the musculature in a characteristic fashion, implying a functional role for the different isoforms (IO, 31). Indeed, the shortening velocity of single skeletal muscle fibers has been shown to be related to both the MHC (24,26) and MLC (12,26) composition of the fiber. Type I MHC has been generally associated with slowtwitch fibers and is thought to have a slower adenosinetriphosphatase (ATPase) activity, whereas the type IIa
THE CONTRACTILE
0363-6143/91
$1.50 Copyright
and IIb MHCs are found in faster contracting fibers. The phenotypic expression of MHC isoforms within a given muscle appears to be under the control of a number of factors; among them developmental (4, 18, 20), neural (5, 25), hormonal (9, 17, 20), and mechanical factors (3, 8, 30). However, little is known regarding the possible interaction between these various regulators of MHC content, or which, if any, predominate over others in the control of MHC expression. Information regarding the degree of cooperativity or competition between those forces that influence MHC isoform distribution promises to greatly enhance our understanding of the overall control of MHC gene expression. Our interest in this study was to contrast two competing influences that have been well characterized with regard to their impact on myosin isoform expression: hypothyroidism and hindlimb suspension. The hormone thyroxine has been shown to be a potent, tissue-specific regulator of MHC gene expression (14,16, 17, 20). Izumo et al. (17) demonstrated that, in slow muscle such as the soleus, hyperthyroidism was associated with an upregulation of the expression of the type IIa MHC messenger RNA, whereas hypothyroidism was associated with an upregulation of type I MHC expression. In fast muscles, hypothyroidism was associated with an increase in type IIa MHC mRNA. These responses to altered thyroid state have also been characterized at the protein level, as Fitzsimons et al. (9) demonstrated that, in slow muscles, hyperthyroidism was associated with an increase in the relative amount of fast myosin, whereas hypothyroidism resulted in a relative increase in the slow myosin content. In addition, slow muscle was found to be much more responsive to alterations in thyroid state than was fast muscle (9). Mechanical factors, such as the load imposed on a muscle, are also thought to have their greatest impact on slow muscles. Removal of weight-bearing activity from slow muscles, as in the hindlimb suspension model (8, 29) or during exposure to a zero gravity environment (3), results in a shift in isomyosin distribution characterized by an increase in the relative amount of the intermediate and the fast myosins (containing the type IIa and IIb MHCs). Conversely, increasing the degree of weight bearing activity through surgical removal of the synergists has been shown to increase the relative amount of slow myosin (containing type I MHC) significantly (30). However, while the effects of hindlimb suspension on myosin isoform distribution have been well character-
0 1991 the American
Physiological
Society
Cl099
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.151.010.035) on October 22, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
Cl100
HYPOTHYROIDISM
AND
SUSPENSION
ized, relatively little is known regarding the impact of suspension on MHC distribution at the mRNA level. Thomason et al. (28) reported that, although type I MHC protein levels had been shown to be decreased following 7 days of suspension, type I MHC mRNA levels did not fall, thus raising the possibility of translational or posttranslational regulation of MHC expression during the course of hindlimb suspension. Given the above, our objectives in this study were to I) determine the relative efficacy of hypothyroidism and hindlimb suspension in the modulation of MHC expression when these two manipulations are imposed simultaneously in an animal and 2) compare the response with these manipulations at both the mRNA level and at the protein level to distinguish between transcriptional and translational control of MHC expression. Here we present evidence that thyroid state predominates over mechanical factors associated with unweighting in the determination of MHC isoform expression. Evidence is also presented suggesting incon gruities in the response to hindlimb suspension of the MHC isoforms vs. their respective mRNA levels. METHODS
Animal care and suspension. Eighteen normal and eighteen thyroidectomized adult female Sprague-Dawley rats (-250 g) were obtained from Taconic Farms (Germantown, NY). Thyroidectomized animals were immediately placed on a regimen of intraperitoneal injections of the antithyroid drug 6-n-propyl-2-thiouracil (PTU, 12 mg/kg body wt) every other day. Animals were housed in temperatureand light-controlled quarters and received food and water ad libitum. All experiments and procedures were approved by the University of California-Irvine Animal Use Committee and conformed to National Institutes of Health guidelines for the humane treatment of animals. Approximately two weeks following the thyroidectomy, the resting metabolic status of the animals was tested to verify an altered thyroid state. Resting oxygen uptake (VO,) was measured according to methods described previously (22). After the determination of metabolic status, animals were assigned to one of four groups: normal control (NC), hypothyroid control (TC), normal suspended (NS), and hypothyroid suspended (TS). Animals in the NS and TS groups were suspended by an .oninvasive procedure that has been described previously (29). Briefly, a tail cast was applied to the animal, leaving the distal one-third of the tail free to allow for proper thermoregulation. The tail cast was attached to a hook at the top of the cage, and the height of the hook was adjusted so that only the front legs could contact the cage floor. This method allows the animals full 360” rotation as well as access to food and water without allowing the hindlimbs to contact the cage floor or walls. Suspension was discontinued and the animal removed from the study if any discoloration of the tail was detected. At the end of 2 wk of hindlimb suspension, the animals were anesthetized with pentobarbital sodium and blood was drawn for plasma 3,5,3’-triiodothyronine (T3) analy-
EFFECTS
IN
SKELETAL
MUSCLE
sis. The animals were then killed with a lethal overdose of pentobarbital sodium, and the heart and the soleus, plantaris, and vastus intermedius (VI) muscles were removed. The heart and the muscles from one leg of each animal were trimmed of connective tissue, weighed, placed into precooled glycerol, and stored at -20°C for subsequent myofibril purification, while the muscles of the other leg were quick frozen and stored at -70°C for RNA extraction. Myofibril isolation and gel electrophoresis. Myofibril purification and protein determination were performed according to a method described previously (27,31). After purification, myofibrils were stored at a protein concentration of 1 mg/ml in storage buffer consisting of 50% glycerol, 100 mM Na4P207, 5 mM EDTA, and 2 mM 2mercaptoethanol (pH 8.8). MHCs were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to a modification of the method described by Danielli-Betto et al. (7). Briefly, the protein sample was mixed with sample buffer that consisted of 62.5 mM tris(hydroxymethyl)aminomethane (Tris), 20% glycerol, 1% 2-mercaptoethanol, 2.3% SDS, and 0.05% bromophenol blue. This mixture was heated at 100°C for 2 min and then -1 pg of protein was loaded onto the gel. The stacking gel was 4% total acrylamide, 2.6% bis-acrylamide (expressed as a percent of the total acrylamide), and 40% glycerol, pH 6.8, whereas the resolving gel was 7% acrylamide, 2.6% bis-acrylamide, and 40% glycerol, pH 8.3. The gels were run at 120 V (constant voltage) for 4-4.5 h until the dye front migrated off of the gel. To determine the cardiac native myosin isoform distribution, the myofibril fraction isolated from the heart was subjected to nondissociating PAGE for 20 h according to the method described previously (27, 31). Both SDS gels and native gels were stained for 1 h in 0.1% Coomassie Blue R-250, 25% methanol, and 8% acetic acid. Destaining occurred by diffusion until the background was clear. After staining and destaining, the gels were scanned densitometrically at 630 nm in a Zeineh soft laser densitometer (BioMedical Instruments, Fullerton, CA) interfaced to an IBM PC computer equipped with software to perform peak area integration analysis. The areas of the peaks corresponding to each band were summated and then the area for each peak expressed as a percent of the total area. In this way, each native myosin or MHC isoform is expressed as a percent of the total myosin or total MHC present on the gel. RNA isolation. Total cellular RNA was isolated from the muscle samples using the guanidium isothiocyanatecesium chloride, ethanol precipitation method (6, 11). The RNA pellet was suspended in diethyl pyrocarbonate pretreated water and stored frozen at -20°C for slot blot analysis. RNA quantities were determined by ultraviolet (UV) absorption at 260 nm. RNA quality was routinely checked by electrophoresis of 0.5-1.0 pugof RNA through a 1% agarose gel in lx Tris-borate-EDTA buffer containing ethidium bromide. The gels were exposed to UV light and checked for the presence of two fluorescing bands, corresponding to the 28s and 18s ribosomal RNA. No evidence of RNA degradation was observed in any of
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.151.010.035) on October 22, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
HYPOTHYROIDISM
AND
SUSPENSION
the samples tested. Preparation of synthetic probes. MHC gene specific oligonucleotides, 20 bases in length, were purchased from the University of California-Irvine Biological Chemistry Department. The sequences of these probes have been reported previously (14). These sequences are located in the 3’-untranslated regions of the MHC isoform mRNAs and have been shown to be highly isoform specific (14). The probes were Y-end labeled with 32P to a specific activity of 4-5 x lo6 cpm/mol by the T4-polynucleotide kinase reaction (21). Labeled oligonucleotides was separated from unlabeled and from unincorporated [T-~~P]ATP by urea PAGE. The probes were repeatedly tested for specificity using Northern blot analysis with both . . 1 . . n * * positive and negative controls for each probe. RNA slot-blot assay. Total RNA (l-2 pg) was blotted onto a Nytran nylon membrane (Schleicher & Schuell, Keene, NH) using a standard slot-blot procedure. The filters were subsequently fixed with UV irradiation. Prehybridization, hybridization, and washing procedures for the blots were all carried out according to the manufacturers recommendations. After the final wash, the membranes were autoradiographed with an intensifying screen at -70°C for l-4 days. mRNA levels were quantified by densitometric scanning of the autoradiograms. The quantified amount of each MHC mRNA in each of the experimental groups was then normalized to the level found in the NC group so that the amount of each MHC mRNA was expressed as a percent change from normal control levels. Plasma T3 determination. Plasma T3 levels in blood samples drawn just prior to death were determined using a commercial radioimmunoassay kit (Gamma Coat [1251] T3, Dade Diagnostics, Cambridge, MA). Statistical analysis. All data are presented as means t SE from several muscles. Data were analyzed using a one-way analysis of variance to determine homogeneity of variance. For each variable measured, data from the experimental groups were tested for significant difference compared with the NC group data. In some instances, tests of significance were also made between data from the TC group and data from the TS group. P values of ~0.05 were considered significant after adjustment using the Bonferroni correction for multiple comparisons (32).
EFFECTS
IN
SKELETAL
Cl101
MUSCLE
1. Evidence for existence of hypothyroid state
TABLE
NC
n Body weight, g Plasma TB, ng/dl Heart weight, mg Cardiac isomyosins, relative % VI
NS
TS
9
9
9
9
299t4
246t4*
