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Effect of thyroid hormone on protein turnover in cultured cardiac myocytes
J Mol Cell Cardiol 17, 897-905 (1985)
Effect o f Thyroid H o r m o n e on Protein Turnover in Cultured Cardiac M y o c y t e s William J. Carter, Wieke S. van der Weijden Benjamin and Fred H. Faas
Veterans Administration Medical Center and Department of Medicine, University of Arkansasfor Medical Sciences, Little Rock, AR 72205, USA (Received5 March 1984, acceptedin revisedform 3 December 1984) w . J . CARTER, W. S. VAN DER WEIJDEN BENJAMINAND F. H. FAAS. Effect of Thyroid Hormone on Protein Turnover in Cultured Cardiac Myocytes. Journalof Molecularand CellularCardiology(1985) 17, 897 905. Since systemic actions of thyroid hormone increase cardiac work, direct effects ofT s on myocardial protein turnover may be obscured in the intact animal. For this reason, the effects ofT 3 on synthesis and degradation of cellular protein were measured in replicate cultures of cardiac myocytes obtained from chick embryos. During the first 3 days of exposure, 10-s M T 3 increased the fractional rate of protein synthesis 10% to 16% and the fractional rate of cell growth 20% to 40% with no change in protein degradation. During the fourth and fifth days of 10 -8 M T 3 exposure, fractional synthesis rates in T 3 cultures increased 15% to 19% but fractional degradation rates also increased 17% to 29% so that growth rates in T s cultures fell to control levels. Similar changes in myocardial protein turnover have occurred in response to T 3 treatment in intact animals. T 3 treatment caused a disproportionately large increase in the rate of myosin heavy chain turnover when compared to total cellular protein and actin. This may be related to the change in amounts of myocardial isomyosins occurring in response to thyroid hormone treatment. KEY WORDS: T 3 ; Triiodothyronine; Thyroid hormone; Cardiac myocytes; Protein turnover; Myosin heavy chain ; Isoenzymes of myosin.
Introduction A d m i n i s t r a t i o n of t r i i o d o t h y r o n i n e (T3) to rats for 7 days c a u s e d m y o c a r d i a l h y p e r t r o p h y w h i c h was a c c o m p a n i e d by a n i n c r e a s e d r a t e of m y o c a r d i a l p r o t e i n synthesis w i t h no c h a n g e in p r o t e i n d e g r a d a t i o n [3]. O n the o t h e r h a n d , the s a m e T 3 t r e a t m e n t i n c r e a s e d p r o t e i n synthesis a n d d e g r a d a t i o n in skeletal m u s c l e w i t h u n c h a n g e d o r d e c r e a s e d skeletal m u s c l e mass [3]. S i n c e T 3 a d m i n i s t r a t i o n in vivo causes m e t a b o l i c effects w h i c h i n c r e a s e c a r d i a c w o r k [9], it is n o t c l e a r w h e t h e r t h e d i f f e r e n t responses o f skeletal a n d c a r d i a c m u s c l e to T 3 are d u e to s e c o n d a r y effects t h a t i n c r e a s e c a r d i a c work, o r to d i f f e r e n t d i r e c t effects o f T 3 on the c a r d i a c m y o c y t e . T o h e l p a n s w e r this q u e s t i o n , the effect o f T 3 on t o t a l p r o t e i n t u r n o v e r has b e e n i n v e s t i g a t e d in t h e c a r d i a c m y o c y t e cell c u l t u r e system. F u r t h e r m o r e , t h y r o i d status a p p e a r s to i n f l u e n c e the
r a t i o o f m y o s i n h e a v y c h a i n v a r i a n t s f o u n d in ventricular muscle and myosin ATPase activity in several species [11, 17]. F o r this reason, the t u r n o v e r o f the m y o s i n h e a v y c h a i n fraction was c o m p a r e d to a c t i n a n d total c e l l u l a r p r o t e i n in c a r d i a c m y o c y t e cultures g r o w n in the p r e s e n c e a n d absence o f t 3 . A c a r d i a c m y o c y t e system d e r i v e d f r o m chick e m b r y o h e a r t cells has b e e n selected for this s t u d y [12]. T h i s system uses a serum-free, hormone supplemented medium that minimizes f i b r o b l a s t o v e r g r o w t h [12]. F u r t h e r m o r e , these cells r e s p o n d to t h y r o i d h o r m o n e by i n c r e a s i n g the u p t a k e o f glucose a n d glucose a n a l o g u e s [14], b y i n c r e a s i n g l a c t a t e p r o d u c t i o n [12], a n d b y i n c r e a s i n g R N A a n d p r o t e i n synthesis [13]. A s t u d y o f T 3 effects o n p r o t e i n t u r n o v e r in this c a r d i a c m y o c y t e system shows a p a t t e r n o f response similar to t h a t o b s e r v e d in vivo. I n i t i a l T 3 t r e a t m e n t
Correspondence should be addressed to: William J. Carter, John L. McCllellan Memerial Veterans' Hospital, 4300 West 7th Street (151-JLM), Little Rock, AR72205, USA. 0022-2828/85/090897 + 09 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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increased protein synthesis and growth with no change in protein breakdown while further T 3 exposure increased protein synthesis and degradation thereby reducing the growth rate toward control levels. In addition, T 3 treatment increased myosin heavy chain turnover to a greater degree than that of actin or total cellular protein. Materials and Methods
Materials Glasgow minimal essential medium, 10% lactalbumin hydrolysate, chicken serum, horse serum and fetal bovine serum were obtained from the Grand Island Biological Company, Grand Island, NY. A lyophilized mixture of insulin, transferrin, and selenium ('ITS premix'), dexamethasone, and fibronectin were purchased from Collaborative Research, Lexington, MA. Rabbit actin, chicken myosin, HEPES*, BES*, TES* and 3,5,3'-triiodo-L-thyronine (T3) were purchased from Sigma Chemical Company, St Louis, MO. Trypsin, 1:250, and tryptose phosphate broth were obtained from Difco Laboratories, Detroit, MI. L-U[14C] leucine, specific activity 342/tCi/#mol, was purchased from New England Nuclear Corporation, Boston, MA and L-4, 513H] leucine, specific radioactivity, 55 mCi/#mol, from ICN Radioisotope Division, Irvine, CA.
Preparation of myocyte cultures The culture medium [12] was Glasgow M E M which contained 100 units penicillin, 100/~g streptomycin, 0.25 #g amphotericin, 5 pg insulin, 5 pg transferrin, and 5 mg selenium/ ml. The medium also contained 8 mM HEPES; 5 mM BES, 5 mM TES, and 10 .7 M dexamethasone. Tryptose phosphate broth, 100 ml, and 10% lactalbumin hydrolysate, 50 ml, were added to each litre of medium and the bicarbonate concentration adjusted to provide a pH of 7.4 in a 5% CO 2 atmosphere. Cardiac ventricles from three dozen 10-day chick embryos were quickly removed, cut into fragments, and rinsed three times in phos-
phate buffered saline (PBS) at 37~ The fragments were gently stirred in 10 ml PBS containing 0.025% trypsin for five consecutive 5 min periods at 37~ using a magnetic stirrer. Supernatants from the first two trypsin treatments were discarded but the final three supernatants which contained dissociated myocytes were placed in centrifuge tubes containing 10 ml of medium supplemented with 25% horse serum. After the cells were pelleted by centrifugation at 300 g for 5 min, this medium was discarded and the cells resuspended in medium containing 5% chicken serum to a standard concentration of 106 cells/ml. Samples (0.5 ml) of this cell suspension were added to 2 ml serum-free medium in 35 m m plastic culture dishes. The culture medium was changed daily and the cells refed with 2 ml serum-free medium per dish. The cells were grown in a 5% CO/-air atmosphere at 37~ When added, sufficient T 3 was dissolved in 10 m g N a O H so that addition of 1 #1 per ml of medium yielded the desired concentration. Control cultures received the same amount of N a O H .
Measurement ofprotein synthesis in myocyte cultures A dual isotope method was used [5]. Myocytes were grown in medium containing 0.1 #Ci [14C] leucine per ml for 5 days to achieve isotopic equilibrium between [14C] leucine in the medium and cellular protein. A 4 h pulse of [3H] leucine, 2 #Ci per ml, was then added. After this pulse, the cells were rapidly harvested and samples of the medium and cell protein were taken for measurement of [14C] and [3HI radioactivity. The fractional rate of cellular protein synthesis per 4 h was calculated as follows [5]. [3H] d/min protein [14C] d/min protein -9 F [3H]/min medium leucine / [14C] d/min medium leucine [3HI d/min protein
-
d- min
* HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; *BES N,N-bis-[2-hydroxyethyl]-2-aminoethanesulfonic acid; *TES N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonicacid.
T 3 and Myocyte Protein Turnover
To measure radioactivity in cell protein, the medium was removed, the cells washed three times with cold PBS, three times with cold 10% trichloroacetic acid (TCA), and the protein residue solubilized in 0.5 M NaOH. An aliquot was counted in a Packard Tricarb scintillation counter set to discriminate [,3HI and [14C] radioactivity. To measure the radioactivity of leucine in the medium, a 1 ml sample was acidified and passed over a 1 x 3 cm column of AG 50 W x 8 resin (Bio Rad) in the acid form. The column was washed with 50 ml water and the amino acids eluted with 2 ml 25% NH4OH. The N H 4 O H was removed by evaporation, the residue dissolved in a small amount of water and counted as described above. The efficiency of [-3HI and [14C] counting was determined by internal standards.
Measurement of protein degradation in myocyte cultures Fractional rates of myocyte protein breakdown were measured as previously described [1]. Myocyte cultures were grown in medium containing 1.0/~Ci [3HI leucine/ml for 5 days. At this time the cells were washed four times with 2 ml samples of medium containing 5 mM unlabelled leucine. The cells were incubated for 4 h and the medium again replaced with fresh medium containing 5 mM leucine. After a final 24 h incubation, the medium was removed, the cells washed three times with cold PBS, three times with cold 10% TCA, and the protein residue dissolved in 0.5 M N a O H . T C A soluble and insoluble counts in the medium were measured. The fractional rate of myocyte protein degradation/24 h was calculated as follows [1]. T C A soluble [3H] c/min in medium [3H] c/min in cellular protein + T C A insoluble [3H] cpm in medium
Isolation of contractileproteins from total cellularprotein Cultures were washed three times with cold PBS and the protein residue from six to eight 35 mm plates was suspended in 2 ml low salt buffer containing 1% mercaptoethanol and 1% sodium dodecyl sulfate and solubilized for
899
electrophoresis by heating to 100~ for l0 rain [5]. The solubilized cell proteins were separated on a one dimensional 7.5% acryla, mide slab gel with a 6 0 : 1 cross link ratio [16]. The gels were fixed and stained with Coomassie Blue R, then destained and stored in 7% acetic acid.
Extraction and determination of protein radioactivity in acrylamide gels A previously described procedure was used [10]. Stained bands were cut from the gel and hydrolyzed overnight at 105~ in 5 ml 6 n HC1 containing 1 #1 mercaptoethanol/ml. The hydrolysate was allowed to stand at - 2 0 ~ for 12 h and the precipitate removed by centrifugation. After the HCI was removed by repeated evaporation, the residue was dissolved in a small volume of water and counted in a Packard Tricarb scintillation counter set to discriminate [3HI and [t4C] counts. The fractional synthesis rates of individual proteins were estimated by comparing [3H]/[-14C] ratios in the protein bands to the [3H]/[14C] ratio of medium leucine as described above.
Protein determination in myocyte cultures After the medium was removed, the cell films were washed with cold PBS and fixed with cold 10% TCA. The washed protein residues were then dissolved in 0.5 M N a O H and the protein content measured using a modified Lowry procedure [-8]. Results
Appearance and activity of cultured cardiac myocytes By 24 h many cells had attached to the plate and put out cytoplasmic extensions. By 48 to 72 h most cells had recovered from the trypsin treatment with disappearance of coarse blebs and vacuoles and approximately 80% showed spontaneous contractions. After 5 days in culture, sheets of synchronously contracting cells were evident. As had been previously noted [12], addition of 10 -1~ to 10 -8 M T 3 did not change the morphology or contractile activity of the cultured cells during the 5-day period o f t 3 exposure.
W. J. Carter et al.
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T A B L E 1. Effect of 4 days exposure to T 3 on myocyte protein content T3
concentration
#g protein 35 m m dish
Control 10 - a ~ M 5 X 10 - l ~ M
205 219 223 232 236 251
10 - 9 M
5 X 10 . 9 M 10 - s ~
AT 3
___5.1
• 3.7* + _ 4_
6.8%
2.9** 4.8** 2.7*** 3.8***
8.8% 13.2% 15.1% 22.4%
Myocyte cultures were incubated for 4 days with the designated T 3 concentrations. T 3 was first added 48 h after the cells were plated. At the end of the experimental period, myocytes were washed with cold PBS, fixed in cold 10% TCA, and dissolved in 0.5 M NaOH for protein determination. Values presented in the Table are means __ S.E.M.of 10 replicate cultures in each group. * P < 0.05, **P < 0.01,***P < 0.001 when compared to the control group by Student's t-test.
Dose response relationship of 7-3 effects on protein content of cultured cardiac myocytes As i n d i c a t e d i n T a b l e 1, 4 d a y s e x p o s u r e to 10 - l ~ to 10 - 8 ~ T 3 c a u s e d a 6 . 8 % to 2 2 . 4 % i n c r e a s e i n t h e p r o t e i n c o n t e n t o f m y o c y t e cultures.
10-SM 3-3
300 ,t i #
250 J
-g 20o
,/
o
& 15o
IO0
As s h o w n i n F i g u r e 1, 10 - 8 M T 3 a d d e d to m y o c y t e s a f t e r 48 h i n c u l t u r e c a u s e d a 1 6 % to 2 1 % i n c r e a s e i n p r o t e i n c o n t e n t d u r i n g t h e subsequent 6 days of exposure. The increment in the rate of growth of T a treated cultures o c c u r r e d d u r i n g t h e first 2 to 4 d a y s o f T 3 e x p o s u r e . F r o m t h e f o u r t h to t h e s i x t h d a y s o f T 3 exposure, growth rates in control and T 3 cultures were approximately equal.
Time course of the effect of T 3 on the content and specific radioactivity of cellular protein of myocytes grown in 14 C leucine labelled medium
50
I Cells Noted
Time course of the effect of T 3 on the protein content of cultured cardiac myocytes
l T~ added
2 4 6 Days T3 exposure
FIGURE i. Time course of the effect of 10 -8 ~t T 3 on the protein content of cultured cardiac myocytes. 9 9 control myocytes; 9 1 4 9 T 3 treated myocytes. T 3 was added 48 h after the ceils were plated. Each data point represents the mean • s.E.~, of eight culture dishes.
To apply the dual isotope method of measuri n g t h e f r a c t i o n a l r a t e o f cell p r o t e i n s y n t h e s i s , it is n e c e s s a r y to s h o w t h a t T 3 does n o t prevent attainment of isotopic equilibrium in cell p r o t e i n o f m y o c y t e s d u r i n g t h e 5 d a y equilibration period. Figure 2 shows the p r o t e i n c o n t e n t a n d specific r a d i o a c t i v i t y o f c e l l u l a r p r o t e i n o f m y o c y t e s g r o w n in m e d i u m
T 3 and Myocyte Protein content 300
250 200
/
/ jr/
Protein
/
15.0 12.5 IO.O
901
Time course of the effect of T 3 on thefractional rate of total protein synthesis, degradation, and growth in cultured cardiac myocytes
Specific rodioocfivit
/
Turnover
% •
.E_ o
Fractional rates of protein synthesis [5] and degradation [ I ] were measured in replicate ~5.0 n~ cultures as indicated in the Materials and ::k IO0 Q) 5O Methods section. Calculated fractional 2.5 growth rates were obtained by subtracting i i i i ~ , i 4 6Cell s 14C Le2u 4 Cells T*3 2 degradation rates from synthesis rates. plated odded plated odded Days T3 exposure O~ys 14C exposure Observed growth rates were determined by harvesting eight replicate cultures at the F I G U R E 2. T i m e course of the effect of 10 - s M T 3 on beginning of the fractional protein synthesis the c o n t e n t a n d specific r a d i o a c t i v i t y of cellular protein and degradation measurements and eight repin m y o c y t e cultures g r o w n in m e d i u m c o n t a i n i n g [14C] leucine. 9 - 9 control m y o c y t e s ; 9 . . . . 9 T3 licate cultures 24 h later. The observed t r e a t e d myocytes. Both protein c o n t e n t and specific growth rate is the percentage increase in the r a d i o a c t i v i t y m e a s u r e m e n t s w e r e m a d e on the s a m e mean protein content of the cultures during g r o u p of control a n d T 3 treated cultures. T 3 and 0.05 ,uCi this interval. Table 2 shows fractional synthe[ x 4 c ] l e u c i n e / m l were a d d e d 72 h after the cells w e r e plated. E a c h d a t a point represents the m e a n __ S.E.M. of sis, degradation, and growth rates after 1 to 5 eight c u l t u r e dishes. days exposure to 10 -8 M T3 and 4 days exposure to 5 x 10 - l ~ M T 3. During the first 3 containing 0.05 #Ci [14C] leucine/ml. As days of 10 -8 M T 3 treatment, the fractional indicated, 10 -8 T 3 did not influence the rate synthesis rates in Ta cultures increased 10% of increase in the specific radioactivity of cel- to 16% with little or no change in fractional lular protein or the attainment of a plateau in degradation rates thereby causing a 20% to the specific radioactivity after 5 days in the 40% increase in fractional growth rates. labelled medium. On the other hand, 10 - 8 M During the fourth and fifth days of 10 - 8 M T 3 T 3 caused the expected 12% to 20% exposure, fractional synthesis rates in T a culincrement in the protein content of the tures continued to be increased by 15% to myocyte cultures. Therefore, 10 .8 M T3 did 19% but fractional degradation rates also not alter attainment of isotopic equilibrium in increased t7% to 29% which caused growth cardiac myocytes grown in medium contain- rates in T 3 cultures to fall to control levels by ing labelled leucine. the fifth day. Therefore, the first 3 days of o
I50
z5
~,
TABLE 2. Effect ofT 3 on fractional rate of total protein synthesis, degradation, and growth in cultured heart cells Fractional rate (%/24 h) of total Length T 3 exposure and T 3 concentration 24 h 48 h 72 h 72 h 96 h 120 h 120 h 96h
(10 -s M) (10 -s M) (10 -s M) (10 - s M) (10 - s M) (10 - s M) (10 -s M) (5 x lo-lO M)
Synthesis
Degradation
C
T3
C
60.8 _+ 0,84 57.0 • 0.88 59.2 _+ 0.68 74.6 -t- 1,4 63.2 __. 1.4 54.6 --_ 0.50 56.4 + 0.62 51.8+0.63
67.9 __ 0.66*** 65.9 __ 0.45*** 67.4 • 0.86*** 81.4 • 1.6"* 75.0 • 1.4"** 62.6 • 0.73*** 65.8 -t- 0,82*** 57.0-t-0.41"**
39.9 + 0.32 39.3 _+ 0.30 41.0 • 0.52 47.0 _+ 0.38 41.5 • 0,57 35.5 • 0.26 43.8 • 0,63 32.4+0.34
Growth (Cal) Ta
40.1 • 40.8 • 42.4 • 48.0 • 50.2 • 45,9 • 51.1 • 34.6•
protein
0.53 0.42 0.34* 0.34 0.54*** 0.64*** 0.44***
Growth (Obs)
C
Ta
C
Ta
20.9 17.7 18.2 27.6 21.7 19.1 12,6 19.4
27.8 25.1 25.0 33.4 24.8 16.7 14.7 22.4
19,8 15.7 17.0 25.4 18.0 16.0 8.9 --
25.4 24.7 24.0 35.3 24.0 13.4 9.9
For protein synthesis measurements, eight myocyte cultures were labelled to equilibrium by adding 0.1 #Ci [14C] leucine/ml of medium for 5 days before the 4 h [3H] leucine pulse. For protein degradation measurements, eight replicate cultures were labelled for 5 days by adding 1 ,uCi [3H] leucine/ml of medium followed by a 24 h chase in medium containing 5.0 mM unlabened leucine. Labelled medium was first added 48 h after the cells were plated. T 3 was added from 1 to 5 days before beginning the [SH] pulse labelling or chase procedure until the cells were harvested. All values for synthesis and degradation rates are means -t-_S.E.M. of eight dishes. The calculated fractional growth rate (cal) was obtained by subtracting the degradation rate from the corresponding synthesis rate. The observed growth rate (obs) was obtained by harvesting eight replicate cultures at the beginning and end of the 24 h chase period and determining the percentage increase in the mean protein content during this interval. * P < 0.05, **P < 0.01, ***P < 0.001 when compared to corresponding control group by Student's t-test.
902
W . J . Carter et aL
10 - s M T a exposure increased the rate of cellular protein synthesis with no increase in degr a d a t i o n ; however, longer exposure to T 3 increased both synthesis and d e g r a d a t i o n of cellular protein. T h e increase in protein degr a d a t i o n was not accompanied by a change in a p p e a r a n c e or contractile activity of the cultured myocytes. Although exposure to 5 x 10 - x ~ M T a for 4 days produced a smaller increase in protein synthesis and d e g r a d a t i o n t h a n 10 . 8 M T a , the lower T 3 c o n c e n t r a t i o n p r o d u c e d a similar p a t t e r n of increased sy nthesis a n d d e g r a d a t i o n of cellular protein. As shown, calculated rates of protein growth derived from measurements of synthesis and d e g r a d a t i o n corresponded well with actual protein a c c u m u l a t i o n observed in replicate cultures.
SDS polyacrylamide gel electrophoresis of total cellular protein obtained from cardiac myocytes grown in 10-8 M T 3 and 5%fetal bovine serum It is i m p o r t a n t to determine whether the increased protein c o n t e n t of T 3 treated cultures results from increased growth of myocytes or c o n t a m i n a t i n g fibroblasts. For this reason, the effects of T 3 and fetal bovine serum on the fibroblast content of myocyte cultures were compared. T o t a l cellular protein obtained from pooled replicate cultures of myocytes that were untreated, grown in 10 -8 M T 3 for 4 days, or grown in 5% fetal bovine serum for 4 days was subjected to SDS polyacrylamide gel electrophoresis. At the time of harvest the protein content of control cultures was 235 • 2.4 #g per 35 m m dish, T a cultures, 280 + 3.1, a n d serum cultures, 790 • 13.0 (mean + S.E.M,, n = 8). T h e fibronectin b a n d was used as a fibroblast m a r k e r a n d the myosin heavy chain b a n d as a myocyte marker [5]. There was no a p p a r e n t difference in the relative density of the myosin heavy chain and fibronectin b a n d s from control a n d T 3 cultures while the density of the fibronectin b a n d from serum cultures was greatly increased. Figure 3 is a densitometer tracing of the myosin heavy chain a n d fibronectin region of the gel. T h e relative size of the fibronectin a n d myosin peaks was not different in control a n d T 3 cultures even though
$
c
FIB
MYO
FIGURE 3. Densitometer tracings of the fibronectin and myosin heavy chain region of an SDS polyacrylamide gel electrophoresis preparation of total cellular protein from myocytes grown in the presence of 10.8 M T 3 and 5% fetal bovine serum. FIB indicates the fibronectin peak and MYO, the myosin heavy chain peak. The C panel refers to control myocytes, the T-3 panel to T s treated myocytes, and the S panel, to serum treated myocytes. T 3 and serum were added to the cultures 48 h after the cells were plated and treatment was continued for 4 days before harvest. the protein content of T 3 treated cultures was increased 20%. This suggests that T 3 did not increase the protein c o n t e n t of the cultures by increasing the growth of c o n t a m i n a t i n g fibroblasts. O n the other h a n d , there was a marked increase in the fibronectin peak in the serum treated cultures where extensive fibroblast overgrowth occurred.
Comparison of the T 3 effect on the fractional synthesis and degradation rates of total ceUular protein and specific contractile proteins T a b l e 3 shows the results of nine experiments in which 32 replicate myocyte cultures were equally divided into control and T 3 groups a n d treated with 10 -8 M T 3 for 5 days before the measurements were made. O n e - h a l f of the cultures from each group were processed individually to determine the synthesis rate of
T 3 and Myocyte Protein Turnover
903
TABLE 3. Comparison of the fractional synthesis rates of total cell protein and actin and myosin heavy chain fractions with the protein content of actin and myosin heavy chain fractions Fractional synthesis rate (% per 24 h) in: Total cell protein
Actin
Myosin H.C.
C
T3
ATa(% )
C
T3
AT3(% )
58.0
63.7
10.3 _+ 1.8
46.1
52.6
14.4 + 2.5
Equilibrium label content (14C leu c/rain) in protein band ---119 164 39.0 _+ 6.9**
C
T3
ATa(% )
64.9
79.9
23.9 _+ 3.0*
51.0
57.0
13.3 4- 7.6
Beginning48 h after plating, myocyte cultures were incubated for 5 days in the presence of 10- 8 MT 3 and 0.1 /ICi [14C] leucine/ml. After the 4 h [3H] leucine pulse, replicate cultures were harvested for measurement of protein synthesisin total cell protein and actin and myosin heavy chain fractions. The dual isotope technique described in the Materials and Methods section was used to measure fractional synthesisrates in the various fractions. Since myocyteswere grown in the presence of [1'*C] leucine long enough for all leucine residues in cell proteins to have the same specificradioactivity as medium leucine, the 14C c/min in actin and myosin heavy chain bands was assumed to be proportional to the protein content of these bands. Values presented are means of nine separate experiments. In each experiment, the fractional synthesisrate of total cell protein is the mean of eight separate dishes while the fractional synthesisrate of actin and myosin heavy chain is derived from eight replicate dishes which were pooled and fractionated by electrophoresis. The percentage T 3 effect is the mean 4- S.E.M.of the effect observed in nine paired experiments. * P < 0.05 and 0.01 when compared to T 3 effect on actin fraction and whole cell protein respectively. **P < 0.05 when compared to T 3 effect on myosinH.C. fraction. Student's t-test used in comparisons.
total cellular protein while one half were pooled, subjected to electrophoresis, and synthesis rates in actin and myosin heavy chain fractions determined. T a t r e a t m e n t increased the fractional rate of total cellular protein synthesis 10.3 • 1.8%, myosin h e a v y chain synthesis 23.9 ___ 3.0%, a n d actin synthesis 14.4 • 2.5% (mean _• S.E.M.). Therefore, T a exposure appears to increase myosin heavy chain synthesis to a greater degree t h a n total cell protein or actin. Since myocytes in each e x p e r i m e n t were grown in m e d i u m containing [14C] leucine for 5 days, all leucine residues in cellular protein are expected to have the same specific activity as m e d i u m leucine [5]. Therefore the [14C] cOntent of the actin a n d myosin heavy chain b a n d s would be expected to be proportional to the protein c o n t e n t of these bands. As shown in T a b l e 3, T 3 exposure increased the [14C] c / m i n 39.0 • 6.9% in actin b a n d s a n d 13.3 ___ 7.6% in myosin heavy chain bands ( m e a n • S.E.M). Since T 3 t r e a t m e n t caused a greater increase in myosin heavy chain synthesis t h a n in actin synthesis, yet resulted in less a c c u m u l a t i o n of myosin heavy chain protein, T a appears to increase the degradation of myosin heavy c h a i n protein to a greater degree t h a n actin.
Discussion
T h e lowest c o n c e n t r a t i o n that consistently increased p r o t e i n a c c u m u l a t i o n in cultured chick embryo heart cells was 10 - l ~ M Ta while 10 - s M T a produced a 22.4% effect (Table 1). E q u i l i b r i u m dialysis has shown that T a in the protein-free m e d i u m used in the present study exists completely in the free form [15]. T h e lowest effective T a concentration in the present study is not greatly different from the 4 x 10 - l ~ M T 3 level that was measured in 10-day chick e m b r y o serum [6]. T h e dose response relationship of T 3 stimulation of protein a c c u m u l a t i o n is similar to that of T 3 stimulation of 2-deoxy-D-glucose uptake that was previously noted in chick e m b r y o heart cells [15]. T h e usefulness of the chick embryo cardiac myocyte system as a model to study thyroid h o r m o n e effects is supported by the observation that the activity of T 3 analogues in this system closely parallels their activity in m a m m a l s in vivo [15]. T h e time course of the effect of 10-8 M T3 on protein a c c u m u l a t i o n in the myocyte cultures showed that the rate of protein a c c u m u lation in T 3 cultures was greater than control d u r i n g the first 4 days o f T 3 exposure but was approximately equal to the control rate
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W . J . Carter et aL
during the fourth to sixth days o f T a exposure (Figs 1 and 2). An analysis of the time course of the effect of 10 - s M T 3 on the rate of protein synthesis and degradation in the myocyte cultures (Table 2) provides an explanation for this observation. During the first 3 days of exposure, T 3 increased the rate of myocyte protein synthesis with little change in protein degradation resulting in a 20% to 40% increase in the rate of myocyte protein growth. After 4 to 5 days of Ts exposure, myocyte protein synthesis remained elevated but protein degradation increased as well so that the rate of growth of T 3 cultures had returned to the control level by the 5th day of T 3 exposure. Although the time frame of events is shorter in the cultured myocytes, a similar pattern of growth has been observed in intact hearts in vivo. When rats were given 20 /~g T3/100 g body weight daily for 7 days, the rate of myocardial protein synthesis increased with no change in the rate of protein breakdown [3-]. A similar increase in myocardial protein synthesis without increased protein degradation occurred in dogs given 0.5 mg T4/kg body weight daily for 7 days [2]. However, during the second week of T 4 treatment, rates of protein synthesis remained elevated but protein degradation rates also increased so that no further growth in cardiac muscle mass occurred [2]. Therefore, the pattern of early growth caused by increased protein synthesis with no change in degradation followed by a later period of increased synthesis balanced by increased degradation occurs in both cultured myocytes and intact hearts in vivo. Caution must be exercised in comparing the effects of T 3 on immature heart cells in vitro to T 3 effects on adult heart muscle in vivo. Although the contractile activity and morphology of the cultured myocytes did not change during the 5 days of T 3 exposure, the increased protein degradation observed in the cultured myocytes after 3 to 4 days of T 3 treatment may be secondary to the metabolic stress o f T 3 on myocytes grown in an artificial environment. Furthermore, cultured heart cells have much greater rates of growth and fractional protein synthesis than intact hearts of adult animals [5]. Therefore increased protein synthesis and degradation following T 3 treatment of cultured myocytes m a y not be
equivalent to similar changes following T s treatment in vivo. T 3 treatment in rats at 20 ktg per 100 g daily for 7 days affected heart and skeletal muscle protein turnover differently [3]. In heart muscle, protein synthesis and protein mass increased without any change in degradation while in skeletal muscle, both protein synthesis and degradation increased with no change in protein mass. Since T 3 treatment in vivo causes systemic changes which increase cardiac work [-9], it appeared that these secondary effects of T 3 might prevent an early direct T 3 stimulation of myocardial protein degradation. However, the fact that isolated cardiac myocytes also respond to T 3 with an early period of increased protein synthesis with no change in breakdown suggests that T 3 has a different intrinsic effect on cardiac muscle. This suggests that c~rd!ac hypertrophy in hyperthyroid animals is a primary rather than secondary effect of thyroid hormone. The low ratio of fibronectin to the myosin heavy chain fraction in cellular protein obtained from current myocyte cultures together with microscopic observations suggests that the serum-free medium was effective in preventing fibroblast overgrowth (Fig. 3). On the other hand, addition of 5% fetal bovine serum to the medium caused a marked increase in fibronectin and overgrowth of the cultures by noncontractile cells. In contrast to fetal bovine serum, 10 - s M T 3 addition did not increase the size of the fibronectin peak or alter cell morphology (Fig. 3). Therefore, the increase in the protein content of the cultures caused by T3 exposure appears to be due to stimulation of myocyte rather than fibroblast growth. To confirm that T s influences the turnover of muscle specific proteins and to show whether individual proteins are affected differently, the effect o f T 3 on the turnover of the myosin heavy chain and actin fractions was investigated. 10 -8 M T 3 increased the fractional rate of protein synthesis 24% in the myosin heavy chain fraction, 14% in the actin fraction, and 10% in total cellular protein (Table 3). Therefore, T 3 stimulated the synthesis rate of muscle specific proteins as much or more than total cellular protein. Furthermore, there was less accumulation of myosin
T 3
and M y o c y t e Protein T u r n o v e r
heavy chain protein in T 3 treated cultures when c o m p a r e d to actin, which suggests that T 3 increased the d e g r a d a t i o n of myosin heavy chain protein to a greater degree t h a n actin. E n h a n c e d myosin heavy chain t u r n o v e r in T3 treated cultures m a y be related to thyroid h o r m o n e i n d u c e d changes in the ratio of specific ventricular isomyosins that have been noted in several species [11, 17]. Since structural differences in v e n t r i c u l a r isomyosins reside in the heavy c h a i n fraction [11, 17], accelerated t u r n o v e r of the myosin heavy chain fraction would facilitate the replacem e n t of one isomyosin with another. T h e e n h a n c e d myosin heavy chain t u r n o v e r was not associated with a p p a r e n t changes in myocyte contractility. I n contrast to the rat heart, only one myosin b a n d was found in the chicken heart by native gel electrophoresis a n d it was not influenced by thyroid status [4]. Yet T 3 ,
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treatment m a y change the myosin molecule without i n f l u e n c i n g the m i g r a t i o n of the myosin b a n d as indicated by the guinea pig heart in which thyroid status influenced myosin A T P a s e activity [7] a n d i m m u n o l o g i c reactivity [4] without altering the electrophoretic mobility of myosin [4]. Nevertheless, the e n h a n c e d myosin heavy chain t u r n o v e r observed in T 3 treated myocytes m a y not indicate a T a - i n d u c e d change in isomyosin content.
Acknowledgements This work was supported by Veterans A d m i n istration research funds, project no. 1308 002, by the N a t i o n a l Institute of Health, g r a n t No. 1-RO 1-AM20718-05, a n d by a g r a n t from the Arkansas C h a p t e r of the A m e r i c a n H e a r t Association. W e acknowledge the able technical assistance ofMax~y E. Lynch.
References l BALLARD,F.J. Regulation of protein accumulation ill cultured cells. BiochemJ 208, 275-287 (1982). 2 BONNIN,C. M., SPARROW,M. P., TAYLOR,R. R. Increased protein synthesis and degradation in the dog heart during thyroxine administration.J Mol Cell Cardiol 15, 245-250 (1983). 3 CARTER,W.j., BENJAMIN,W. S., FAAS,F. H. Effect of hyperthyroidismon protein turnover in skeletal and cardiac muscle as measured by [I*C] tyrosine infusion.BiochemJ 204, 69~74 (1982). 4 CLARK,W. A., CHIZZONITE,R. A., EVERETT,A. W., RA~INOWITZ,M., ZAK, R. Species correlations between cardiac isomyosins.A comparison of electrophoretic and immunologicalproperties.J Biol Chem 257, 5449-5454 (1982). 5 CLARK,W. A., ZAK, R. Assessment of fractional rates of protein synthesis in cardiac muscle cultures after equilibrium labeling.J Biol Chem 256, 4863-4870 (1981). 6 GASPARD,J. K., KLITGAARD,H. M., WONDERaEM,R. Somatomedin and thyroid hormones in the developing chick embryo. Proc Soc Exp Biol Med 166, 24-27 (1981) 7 GOODKIND,M. J., DAMBACn,G. E., TnVRUM,P. T., LucnI, R.J. Effect of thyroxine on ventricular myocardial contractility and ATPase activity in guinea pigs. AmJ Physio1226, 66-72 (1974). 8 HARTRE~;,E. F. A modification of the Lowry method that gives a linear photometric response. Anal Biochem 48, 422-427 (1972). 9 Hown'T, G., ROWLANDS,D. J., Lt~uNo, D. Y. T. et al. Myocardial contractility and effects of beta-adrenergic blockade in hypothyroidismand hyperthyroidism.Clin Sei 34, 485-495 (1968). 10 MARTIN,A. F., PRIOR,G., ZAK,R. Determination of specific radioactivity of amino acids in proteins directly on polyacrylamidegels: an application to L-leucine.Anal Biochem72, 577-585 (1976). 11 MORKIN,E., FLINK, I. L., GOLDMAN,S. Biochemical and physiologic effects of thyroid hormone on cardiac performance. Prog Cardiovasc Dis 25, 435-464 (1983). 12 SCHWARTZ,H., GORDON,A. Effect of triiodothyronine on chick embryo heart cells maintained in tissue culture. IsraelJ Med Sci 11,877 883 (1975). i3 SEGAL,J., GORDON,A. The effects of actinomycin D, puromycin, cycloheximide, and hydroxyurea on 3',5,3triiodo-L-thyronine stimulated 2-deoxy-D-glucoseuptake in chick embryo heart cells in vitro. Endocrinology101, 150-156 (1977). 14 SEGALJ., GORDON,A. The effect of 3',5,3-triiodo-L-thyronine on the kinetic parameters of sugar transport in cultured chick embryo heart cells. Endocrinology101, 1468-1474 (1977). 15 SEOAL,J., SCHWARTZ,H., GORDON,A. The effect of triiodothyronine on 2-deoxy-D-[1-3H] glucose uptake in cultured chick embryo heart cells. Endocrinology101, 143-149 (1977). 16 WEBER,K., OSBORN,M. The reliability of molecular weight determinations by dodecyl sulfate~-polyacrylamide gel electrophoresis.J Biol Chem 244, 4406-4412 (1969). 17 ZAK,R., CHIZZONITE,R. A., EVERETT,A. W., CLARI~,W. A. Study of ventricular isomyosinsduring normal and thyroid hormone induced cardiac growth.J Mol Cell Cardiol 14 [Suppl 3], 111-117 (1982).
Effect o f Thyroid H o r m o n e on Protein Turnover in Cultured Cardiac M y o c y t e s William J. Carter, Wieke S. van der Weijden Benjamin and Fred H. Faas
Veterans Administration Medical Center and Department of Medicine, University of Arkansasfor Medical Sciences, Little Rock, AR 72205, USA (Received5 March 1984, acceptedin revisedform 3 December 1984) w . J . CARTER, W. S. VAN DER WEIJDEN BENJAMINAND F. H. FAAS. Effect of Thyroid Hormone on Protein Turnover in Cultured Cardiac Myocytes. Journalof Molecularand CellularCardiology(1985) 17, 897 905. Since systemic actions of thyroid hormone increase cardiac work, direct effects ofT s on myocardial protein turnover may be obscured in the intact animal. For this reason, the effects ofT 3 on synthesis and degradation of cellular protein were measured in replicate cultures of cardiac myocytes obtained from chick embryos. During the first 3 days of exposure, 10-s M T 3 increased the fractional rate of protein synthesis 10% to 16% and the fractional rate of cell growth 20% to 40% with no change in protein degradation. During the fourth and fifth days of 10 -8 M T 3 exposure, fractional synthesis rates in T 3 cultures increased 15% to 19% but fractional degradation rates also increased 17% to 29% so that growth rates in T s cultures fell to control levels. Similar changes in myocardial protein turnover have occurred in response to T 3 treatment in intact animals. T 3 treatment caused a disproportionately large increase in the rate of myosin heavy chain turnover when compared to total cellular protein and actin. This may be related to the change in amounts of myocardial isomyosins occurring in response to thyroid hormone treatment. KEY WORDS: T 3 ; Triiodothyronine; Thyroid hormone; Cardiac myocytes; Protein turnover; Myosin heavy chain ; Isoenzymes of myosin.
Introduction A d m i n i s t r a t i o n of t r i i o d o t h y r o n i n e (T3) to rats for 7 days c a u s e d m y o c a r d i a l h y p e r t r o p h y w h i c h was a c c o m p a n i e d by a n i n c r e a s e d r a t e of m y o c a r d i a l p r o t e i n synthesis w i t h no c h a n g e in p r o t e i n d e g r a d a t i o n [3]. O n the o t h e r h a n d , the s a m e T 3 t r e a t m e n t i n c r e a s e d p r o t e i n synthesis a n d d e g r a d a t i o n in skeletal m u s c l e w i t h u n c h a n g e d o r d e c r e a s e d skeletal m u s c l e mass [3]. S i n c e T 3 a d m i n i s t r a t i o n in vivo causes m e t a b o l i c effects w h i c h i n c r e a s e c a r d i a c w o r k [9], it is n o t c l e a r w h e t h e r t h e d i f f e r e n t responses o f skeletal a n d c a r d i a c m u s c l e to T 3 are d u e to s e c o n d a r y effects t h a t i n c r e a s e c a r d i a c work, o r to d i f f e r e n t d i r e c t effects o f T 3 on the c a r d i a c m y o c y t e . T o h e l p a n s w e r this q u e s t i o n , the effect o f T 3 on t o t a l p r o t e i n t u r n o v e r has b e e n i n v e s t i g a t e d in t h e c a r d i a c m y o c y t e cell c u l t u r e system. F u r t h e r m o r e , t h y r o i d status a p p e a r s to i n f l u e n c e the
r a t i o o f m y o s i n h e a v y c h a i n v a r i a n t s f o u n d in ventricular muscle and myosin ATPase activity in several species [11, 17]. F o r this reason, the t u r n o v e r o f the m y o s i n h e a v y c h a i n fraction was c o m p a r e d to a c t i n a n d total c e l l u l a r p r o t e i n in c a r d i a c m y o c y t e cultures g r o w n in the p r e s e n c e a n d absence o f t 3 . A c a r d i a c m y o c y t e system d e r i v e d f r o m chick e m b r y o h e a r t cells has b e e n selected for this s t u d y [12]. T h i s system uses a serum-free, hormone supplemented medium that minimizes f i b r o b l a s t o v e r g r o w t h [12]. F u r t h e r m o r e , these cells r e s p o n d to t h y r o i d h o r m o n e by i n c r e a s i n g the u p t a k e o f glucose a n d glucose a n a l o g u e s [14], b y i n c r e a s i n g l a c t a t e p r o d u c t i o n [12], a n d b y i n c r e a s i n g R N A a n d p r o t e i n synthesis [13]. A s t u d y o f T 3 effects o n p r o t e i n t u r n o v e r in this c a r d i a c m y o c y t e system shows a p a t t e r n o f response similar to t h a t o b s e r v e d in vivo. I n i t i a l T 3 t r e a t m e n t
Correspondence should be addressed to: William J. Carter, John L. McCllellan Memerial Veterans' Hospital, 4300 West 7th Street (151-JLM), Little Rock, AR72205, USA. 0022-2828/85/090897 + 09 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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increased protein synthesis and growth with no change in protein breakdown while further T 3 exposure increased protein synthesis and degradation thereby reducing the growth rate toward control levels. In addition, T 3 treatment increased myosin heavy chain turnover to a greater degree than that of actin or total cellular protein. Materials and Methods
Materials Glasgow minimal essential medium, 10% lactalbumin hydrolysate, chicken serum, horse serum and fetal bovine serum were obtained from the Grand Island Biological Company, Grand Island, NY. A lyophilized mixture of insulin, transferrin, and selenium ('ITS premix'), dexamethasone, and fibronectin were purchased from Collaborative Research, Lexington, MA. Rabbit actin, chicken myosin, HEPES*, BES*, TES* and 3,5,3'-triiodo-L-thyronine (T3) were purchased from Sigma Chemical Company, St Louis, MO. Trypsin, 1:250, and tryptose phosphate broth were obtained from Difco Laboratories, Detroit, MI. L-U[14C] leucine, specific activity 342/tCi/#mol, was purchased from New England Nuclear Corporation, Boston, MA and L-4, 513H] leucine, specific radioactivity, 55 mCi/#mol, from ICN Radioisotope Division, Irvine, CA.
Preparation of myocyte cultures The culture medium [12] was Glasgow M E M which contained 100 units penicillin, 100/~g streptomycin, 0.25 #g amphotericin, 5 pg insulin, 5 pg transferrin, and 5 mg selenium/ ml. The medium also contained 8 mM HEPES; 5 mM BES, 5 mM TES, and 10 .7 M dexamethasone. Tryptose phosphate broth, 100 ml, and 10% lactalbumin hydrolysate, 50 ml, were added to each litre of medium and the bicarbonate concentration adjusted to provide a pH of 7.4 in a 5% CO 2 atmosphere. Cardiac ventricles from three dozen 10-day chick embryos were quickly removed, cut into fragments, and rinsed three times in phos-
phate buffered saline (PBS) at 37~ The fragments were gently stirred in 10 ml PBS containing 0.025% trypsin for five consecutive 5 min periods at 37~ using a magnetic stirrer. Supernatants from the first two trypsin treatments were discarded but the final three supernatants which contained dissociated myocytes were placed in centrifuge tubes containing 10 ml of medium supplemented with 25% horse serum. After the cells were pelleted by centrifugation at 300 g for 5 min, this medium was discarded and the cells resuspended in medium containing 5% chicken serum to a standard concentration of 106 cells/ml. Samples (0.5 ml) of this cell suspension were added to 2 ml serum-free medium in 35 m m plastic culture dishes. The culture medium was changed daily and the cells refed with 2 ml serum-free medium per dish. The cells were grown in a 5% CO/-air atmosphere at 37~ When added, sufficient T 3 was dissolved in 10 m g N a O H so that addition of 1 #1 per ml of medium yielded the desired concentration. Control cultures received the same amount of N a O H .
Measurement ofprotein synthesis in myocyte cultures A dual isotope method was used [5]. Myocytes were grown in medium containing 0.1 #Ci [14C] leucine per ml for 5 days to achieve isotopic equilibrium between [14C] leucine in the medium and cellular protein. A 4 h pulse of [3H] leucine, 2 #Ci per ml, was then added. After this pulse, the cells were rapidly harvested and samples of the medium and cell protein were taken for measurement of [14C] and [3HI radioactivity. The fractional rate of cellular protein synthesis per 4 h was calculated as follows [5]. [3H] d/min protein [14C] d/min protein -9 F [3H]/min medium leucine / [14C] d/min medium leucine [3HI d/min protein
-
d- min
* HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; *BES N,N-bis-[2-hydroxyethyl]-2-aminoethanesulfonic acid; *TES N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonicacid.
T 3 and Myocyte Protein Turnover
To measure radioactivity in cell protein, the medium was removed, the cells washed three times with cold PBS, three times with cold 10% trichloroacetic acid (TCA), and the protein residue solubilized in 0.5 M NaOH. An aliquot was counted in a Packard Tricarb scintillation counter set to discriminate [,3HI and [14C] radioactivity. To measure the radioactivity of leucine in the medium, a 1 ml sample was acidified and passed over a 1 x 3 cm column of AG 50 W x 8 resin (Bio Rad) in the acid form. The column was washed with 50 ml water and the amino acids eluted with 2 ml 25% NH4OH. The N H 4 O H was removed by evaporation, the residue dissolved in a small amount of water and counted as described above. The efficiency of [-3HI and [14C] counting was determined by internal standards.
Measurement of protein degradation in myocyte cultures Fractional rates of myocyte protein breakdown were measured as previously described [1]. Myocyte cultures were grown in medium containing 1.0/~Ci [3HI leucine/ml for 5 days. At this time the cells were washed four times with 2 ml samples of medium containing 5 mM unlabelled leucine. The cells were incubated for 4 h and the medium again replaced with fresh medium containing 5 mM leucine. After a final 24 h incubation, the medium was removed, the cells washed three times with cold PBS, three times with cold 10% TCA, and the protein residue dissolved in 0.5 M N a O H . T C A soluble and insoluble counts in the medium were measured. The fractional rate of myocyte protein degradation/24 h was calculated as follows [1]. T C A soluble [3H] c/min in medium [3H] c/min in cellular protein + T C A insoluble [3H] cpm in medium
Isolation of contractileproteins from total cellularprotein Cultures were washed three times with cold PBS and the protein residue from six to eight 35 mm plates was suspended in 2 ml low salt buffer containing 1% mercaptoethanol and 1% sodium dodecyl sulfate and solubilized for
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electrophoresis by heating to 100~ for l0 rain [5]. The solubilized cell proteins were separated on a one dimensional 7.5% acryla, mide slab gel with a 6 0 : 1 cross link ratio [16]. The gels were fixed and stained with Coomassie Blue R, then destained and stored in 7% acetic acid.
Extraction and determination of protein radioactivity in acrylamide gels A previously described procedure was used [10]. Stained bands were cut from the gel and hydrolyzed overnight at 105~ in 5 ml 6 n HC1 containing 1 #1 mercaptoethanol/ml. The hydrolysate was allowed to stand at - 2 0 ~ for 12 h and the precipitate removed by centrifugation. After the HCI was removed by repeated evaporation, the residue was dissolved in a small volume of water and counted in a Packard Tricarb scintillation counter set to discriminate [3HI and [t4C] counts. The fractional synthesis rates of individual proteins were estimated by comparing [3H]/[-14C] ratios in the protein bands to the [3H]/[14C] ratio of medium leucine as described above.
Protein determination in myocyte cultures After the medium was removed, the cell films were washed with cold PBS and fixed with cold 10% TCA. The washed protein residues were then dissolved in 0.5 M N a O H and the protein content measured using a modified Lowry procedure [-8]. Results
Appearance and activity of cultured cardiac myocytes By 24 h many cells had attached to the plate and put out cytoplasmic extensions. By 48 to 72 h most cells had recovered from the trypsin treatment with disappearance of coarse blebs and vacuoles and approximately 80% showed spontaneous contractions. After 5 days in culture, sheets of synchronously contracting cells were evident. As had been previously noted [12], addition of 10 -1~ to 10 -8 M T 3 did not change the morphology or contractile activity of the cultured cells during the 5-day period o f t 3 exposure.
W. J. Carter et al.
900
T A B L E 1. Effect of 4 days exposure to T 3 on myocyte protein content T3
concentration
#g protein 35 m m dish
Control 10 - a ~ M 5 X 10 - l ~ M
205 219 223 232 236 251
10 - 9 M
5 X 10 . 9 M 10 - s ~
AT 3
___5.1
• 3.7* + _ 4_
6.8%
2.9** 4.8** 2.7*** 3.8***
8.8% 13.2% 15.1% 22.4%
Myocyte cultures were incubated for 4 days with the designated T 3 concentrations. T 3 was first added 48 h after the cells were plated. At the end of the experimental period, myocytes were washed with cold PBS, fixed in cold 10% TCA, and dissolved in 0.5 M NaOH for protein determination. Values presented in the Table are means __ S.E.M.of 10 replicate cultures in each group. * P < 0.05, **P < 0.01,***P < 0.001 when compared to the control group by Student's t-test.
Dose response relationship of 7-3 effects on protein content of cultured cardiac myocytes As i n d i c a t e d i n T a b l e 1, 4 d a y s e x p o s u r e to 10 - l ~ to 10 - 8 ~ T 3 c a u s e d a 6 . 8 % to 2 2 . 4 % i n c r e a s e i n t h e p r o t e i n c o n t e n t o f m y o c y t e cultures.
10-SM 3-3
300 ,t i #
250 J
-g 20o
,/
o
& 15o
IO0
As s h o w n i n F i g u r e 1, 10 - 8 M T 3 a d d e d to m y o c y t e s a f t e r 48 h i n c u l t u r e c a u s e d a 1 6 % to 2 1 % i n c r e a s e i n p r o t e i n c o n t e n t d u r i n g t h e subsequent 6 days of exposure. The increment in the rate of growth of T a treated cultures o c c u r r e d d u r i n g t h e first 2 to 4 d a y s o f T 3 e x p o s u r e . F r o m t h e f o u r t h to t h e s i x t h d a y s o f T 3 exposure, growth rates in control and T 3 cultures were approximately equal.
Time course of the effect of T 3 on the content and specific radioactivity of cellular protein of myocytes grown in 14 C leucine labelled medium
50
I Cells Noted
Time course of the effect of T 3 on the protein content of cultured cardiac myocytes
l T~ added
2 4 6 Days T3 exposure
FIGURE i. Time course of the effect of 10 -8 ~t T 3 on the protein content of cultured cardiac myocytes. 9 9 control myocytes; 9 1 4 9 T 3 treated myocytes. T 3 was added 48 h after the ceils were plated. Each data point represents the mean • s.E.~, of eight culture dishes.
To apply the dual isotope method of measuri n g t h e f r a c t i o n a l r a t e o f cell p r o t e i n s y n t h e s i s , it is n e c e s s a r y to s h o w t h a t T 3 does n o t prevent attainment of isotopic equilibrium in cell p r o t e i n o f m y o c y t e s d u r i n g t h e 5 d a y equilibration period. Figure 2 shows the p r o t e i n c o n t e n t a n d specific r a d i o a c t i v i t y o f c e l l u l a r p r o t e i n o f m y o c y t e s g r o w n in m e d i u m
T 3 and Myocyte Protein content 300
250 200
/
/ jr/
Protein
/
15.0 12.5 IO.O
901
Time course of the effect of T 3 on thefractional rate of total protein synthesis, degradation, and growth in cultured cardiac myocytes
Specific rodioocfivit
/
Turnover
% •
.E_ o
Fractional rates of protein synthesis [5] and degradation [ I ] were measured in replicate ~5.0 n~ cultures as indicated in the Materials and ::k IO0 Q) 5O Methods section. Calculated fractional 2.5 growth rates were obtained by subtracting i i i i ~ , i 4 6Cell s 14C Le2u 4 Cells T*3 2 degradation rates from synthesis rates. plated odded plated odded Days T3 exposure O~ys 14C exposure Observed growth rates were determined by harvesting eight replicate cultures at the F I G U R E 2. T i m e course of the effect of 10 - s M T 3 on beginning of the fractional protein synthesis the c o n t e n t a n d specific r a d i o a c t i v i t y of cellular protein and degradation measurements and eight repin m y o c y t e cultures g r o w n in m e d i u m c o n t a i n i n g [14C] leucine. 9 - 9 control m y o c y t e s ; 9 . . . . 9 T3 licate cultures 24 h later. The observed t r e a t e d myocytes. Both protein c o n t e n t and specific growth rate is the percentage increase in the r a d i o a c t i v i t y m e a s u r e m e n t s w e r e m a d e on the s a m e mean protein content of the cultures during g r o u p of control a n d T 3 treated cultures. T 3 and 0.05 ,uCi this interval. Table 2 shows fractional synthe[ x 4 c ] l e u c i n e / m l were a d d e d 72 h after the cells w e r e plated. E a c h d a t a point represents the m e a n __ S.E.M. of sis, degradation, and growth rates after 1 to 5 eight c u l t u r e dishes. days exposure to 10 -8 M T3 and 4 days exposure to 5 x 10 - l ~ M T 3. During the first 3 containing 0.05 #Ci [14C] leucine/ml. As days of 10 -8 M T 3 treatment, the fractional indicated, 10 -8 T 3 did not influence the rate synthesis rates in Ta cultures increased 10% of increase in the specific radioactivity of cel- to 16% with little or no change in fractional lular protein or the attainment of a plateau in degradation rates thereby causing a 20% to the specific radioactivity after 5 days in the 40% increase in fractional growth rates. labelled medium. On the other hand, 10 - 8 M During the fourth and fifth days of 10 - 8 M T 3 T 3 caused the expected 12% to 20% exposure, fractional synthesis rates in T a culincrement in the protein content of the tures continued to be increased by 15% to myocyte cultures. Therefore, 10 .8 M T3 did 19% but fractional degradation rates also not alter attainment of isotopic equilibrium in increased t7% to 29% which caused growth cardiac myocytes grown in medium contain- rates in T 3 cultures to fall to control levels by ing labelled leucine. the fifth day. Therefore, the first 3 days of o
I50
z5
~,
TABLE 2. Effect ofT 3 on fractional rate of total protein synthesis, degradation, and growth in cultured heart cells Fractional rate (%/24 h) of total Length T 3 exposure and T 3 concentration 24 h 48 h 72 h 72 h 96 h 120 h 120 h 96h
(10 -s M) (10 -s M) (10 -s M) (10 - s M) (10 - s M) (10 - s M) (10 -s M) (5 x lo-lO M)
Synthesis
Degradation
C
T3
C
60.8 _+ 0,84 57.0 • 0.88 59.2 _+ 0.68 74.6 -t- 1,4 63.2 __. 1.4 54.6 --_ 0.50 56.4 + 0.62 51.8+0.63
67.9 __ 0.66*** 65.9 __ 0.45*** 67.4 • 0.86*** 81.4 • 1.6"* 75.0 • 1.4"** 62.6 • 0.73*** 65.8 -t- 0,82*** 57.0-t-0.41"**
39.9 + 0.32 39.3 _+ 0.30 41.0 • 0.52 47.0 _+ 0.38 41.5 • 0,57 35.5 • 0.26 43.8 • 0,63 32.4+0.34
Growth (Cal) Ta
40.1 • 40.8 • 42.4 • 48.0 • 50.2 • 45,9 • 51.1 • 34.6•
protein
0.53 0.42 0.34* 0.34 0.54*** 0.64*** 0.44***
Growth (Obs)
C
Ta
C
Ta
20.9 17.7 18.2 27.6 21.7 19.1 12,6 19.4
27.8 25.1 25.0 33.4 24.8 16.7 14.7 22.4
19,8 15.7 17.0 25.4 18.0 16.0 8.9 --
25.4 24.7 24.0 35.3 24.0 13.4 9.9
For protein synthesis measurements, eight myocyte cultures were labelled to equilibrium by adding 0.1 #Ci [14C] leucine/ml of medium for 5 days before the 4 h [3H] leucine pulse. For protein degradation measurements, eight replicate cultures were labelled for 5 days by adding 1 ,uCi [3H] leucine/ml of medium followed by a 24 h chase in medium containing 5.0 mM unlabened leucine. Labelled medium was first added 48 h after the cells were plated. T 3 was added from 1 to 5 days before beginning the [SH] pulse labelling or chase procedure until the cells were harvested. All values for synthesis and degradation rates are means -t-_S.E.M. of eight dishes. The calculated fractional growth rate (cal) was obtained by subtracting the degradation rate from the corresponding synthesis rate. The observed growth rate (obs) was obtained by harvesting eight replicate cultures at the beginning and end of the 24 h chase period and determining the percentage increase in the mean protein content during this interval. * P < 0.05, **P < 0.01, ***P < 0.001 when compared to corresponding control group by Student's t-test.
902
W . J . Carter et aL
10 - s M T a exposure increased the rate of cellular protein synthesis with no increase in degr a d a t i o n ; however, longer exposure to T 3 increased both synthesis and d e g r a d a t i o n of cellular protein. T h e increase in protein degr a d a t i o n was not accompanied by a change in a p p e a r a n c e or contractile activity of the cultured myocytes. Although exposure to 5 x 10 - x ~ M T a for 4 days produced a smaller increase in protein synthesis and d e g r a d a t i o n t h a n 10 . 8 M T a , the lower T 3 c o n c e n t r a t i o n p r o d u c e d a similar p a t t e r n of increased sy nthesis a n d d e g r a d a t i o n of cellular protein. As shown, calculated rates of protein growth derived from measurements of synthesis and d e g r a d a t i o n corresponded well with actual protein a c c u m u l a t i o n observed in replicate cultures.
SDS polyacrylamide gel electrophoresis of total cellular protein obtained from cardiac myocytes grown in 10-8 M T 3 and 5%fetal bovine serum It is i m p o r t a n t to determine whether the increased protein c o n t e n t of T 3 treated cultures results from increased growth of myocytes or c o n t a m i n a t i n g fibroblasts. For this reason, the effects of T 3 and fetal bovine serum on the fibroblast content of myocyte cultures were compared. T o t a l cellular protein obtained from pooled replicate cultures of myocytes that were untreated, grown in 10 -8 M T 3 for 4 days, or grown in 5% fetal bovine serum for 4 days was subjected to SDS polyacrylamide gel electrophoresis. At the time of harvest the protein content of control cultures was 235 • 2.4 #g per 35 m m dish, T a cultures, 280 + 3.1, a n d serum cultures, 790 • 13.0 (mean + S.E.M,, n = 8). T h e fibronectin b a n d was used as a fibroblast m a r k e r a n d the myosin heavy chain b a n d as a myocyte marker [5]. There was no a p p a r e n t difference in the relative density of the myosin heavy chain and fibronectin b a n d s from control a n d T 3 cultures while the density of the fibronectin b a n d from serum cultures was greatly increased. Figure 3 is a densitometer tracing of the myosin heavy chain a n d fibronectin region of the gel. T h e relative size of the fibronectin a n d myosin peaks was not different in control a n d T 3 cultures even though
$
c
FIB
MYO
FIGURE 3. Densitometer tracings of the fibronectin and myosin heavy chain region of an SDS polyacrylamide gel electrophoresis preparation of total cellular protein from myocytes grown in the presence of 10.8 M T 3 and 5% fetal bovine serum. FIB indicates the fibronectin peak and MYO, the myosin heavy chain peak. The C panel refers to control myocytes, the T-3 panel to T s treated myocytes, and the S panel, to serum treated myocytes. T 3 and serum were added to the cultures 48 h after the cells were plated and treatment was continued for 4 days before harvest. the protein content of T 3 treated cultures was increased 20%. This suggests that T 3 did not increase the protein c o n t e n t of the cultures by increasing the growth of c o n t a m i n a t i n g fibroblasts. O n the other h a n d , there was a marked increase in the fibronectin peak in the serum treated cultures where extensive fibroblast overgrowth occurred.
Comparison of the T 3 effect on the fractional synthesis and degradation rates of total ceUular protein and specific contractile proteins T a b l e 3 shows the results of nine experiments in which 32 replicate myocyte cultures were equally divided into control and T 3 groups a n d treated with 10 -8 M T 3 for 5 days before the measurements were made. O n e - h a l f of the cultures from each group were processed individually to determine the synthesis rate of
T 3 and Myocyte Protein Turnover
903
TABLE 3. Comparison of the fractional synthesis rates of total cell protein and actin and myosin heavy chain fractions with the protein content of actin and myosin heavy chain fractions Fractional synthesis rate (% per 24 h) in: Total cell protein
Actin
Myosin H.C.
C
T3
ATa(% )
C
T3
AT3(% )
58.0
63.7
10.3 _+ 1.8
46.1
52.6
14.4 + 2.5
Equilibrium label content (14C leu c/rain) in protein band ---119 164 39.0 _+ 6.9**
C
T3
ATa(% )
64.9
79.9
23.9 _+ 3.0*
51.0
57.0
13.3 4- 7.6
Beginning48 h after plating, myocyte cultures were incubated for 5 days in the presence of 10- 8 MT 3 and 0.1 /ICi [14C] leucine/ml. After the 4 h [3H] leucine pulse, replicate cultures were harvested for measurement of protein synthesisin total cell protein and actin and myosin heavy chain fractions. The dual isotope technique described in the Materials and Methods section was used to measure fractional synthesisrates in the various fractions. Since myocyteswere grown in the presence of [1'*C] leucine long enough for all leucine residues in cell proteins to have the same specificradioactivity as medium leucine, the 14C c/min in actin and myosin heavy chain bands was assumed to be proportional to the protein content of these bands. Values presented are means of nine separate experiments. In each experiment, the fractional synthesisrate of total cell protein is the mean of eight separate dishes while the fractional synthesisrate of actin and myosin heavy chain is derived from eight replicate dishes which were pooled and fractionated by electrophoresis. The percentage T 3 effect is the mean 4- S.E.M.of the effect observed in nine paired experiments. * P < 0.05 and 0.01 when compared to T 3 effect on actin fraction and whole cell protein respectively. **P < 0.05 when compared to T 3 effect on myosinH.C. fraction. Student's t-test used in comparisons.
total cellular protein while one half were pooled, subjected to electrophoresis, and synthesis rates in actin and myosin heavy chain fractions determined. T a t r e a t m e n t increased the fractional rate of total cellular protein synthesis 10.3 • 1.8%, myosin h e a v y chain synthesis 23.9 ___ 3.0%, a n d actin synthesis 14.4 • 2.5% (mean _• S.E.M.). Therefore, T a exposure appears to increase myosin heavy chain synthesis to a greater degree t h a n total cell protein or actin. Since myocytes in each e x p e r i m e n t were grown in m e d i u m containing [14C] leucine for 5 days, all leucine residues in cellular protein are expected to have the same specific activity as m e d i u m leucine [5]. Therefore the [14C] cOntent of the actin a n d myosin heavy chain b a n d s would be expected to be proportional to the protein c o n t e n t of these bands. As shown in T a b l e 3, T 3 exposure increased the [14C] c / m i n 39.0 • 6.9% in actin b a n d s a n d 13.3 ___ 7.6% in myosin heavy chain bands ( m e a n • S.E.M). Since T 3 t r e a t m e n t caused a greater increase in myosin heavy chain synthesis t h a n in actin synthesis, yet resulted in less a c c u m u l a t i o n of myosin heavy chain protein, T a appears to increase the degradation of myosin heavy c h a i n protein to a greater degree t h a n actin.
Discussion
T h e lowest c o n c e n t r a t i o n that consistently increased p r o t e i n a c c u m u l a t i o n in cultured chick embryo heart cells was 10 - l ~ M Ta while 10 - s M T a produced a 22.4% effect (Table 1). E q u i l i b r i u m dialysis has shown that T a in the protein-free m e d i u m used in the present study exists completely in the free form [15]. T h e lowest effective T a concentration in the present study is not greatly different from the 4 x 10 - l ~ M T 3 level that was measured in 10-day chick e m b r y o serum [6]. T h e dose response relationship of T 3 stimulation of protein a c c u m u l a t i o n is similar to that of T 3 stimulation of 2-deoxy-D-glucose uptake that was previously noted in chick e m b r y o heart cells [15]. T h e usefulness of the chick embryo cardiac myocyte system as a model to study thyroid h o r m o n e effects is supported by the observation that the activity of T 3 analogues in this system closely parallels their activity in m a m m a l s in vivo [15]. T h e time course of the effect of 10-8 M T3 on protein a c c u m u l a t i o n in the myocyte cultures showed that the rate of protein a c c u m u lation in T 3 cultures was greater than control d u r i n g the first 4 days o f T 3 exposure but was approximately equal to the control rate
904
W . J . Carter et aL
during the fourth to sixth days o f T a exposure (Figs 1 and 2). An analysis of the time course of the effect of 10 - s M T 3 on the rate of protein synthesis and degradation in the myocyte cultures (Table 2) provides an explanation for this observation. During the first 3 days of exposure, T 3 increased the rate of myocyte protein synthesis with little change in protein degradation resulting in a 20% to 40% increase in the rate of myocyte protein growth. After 4 to 5 days of Ts exposure, myocyte protein synthesis remained elevated but protein degradation increased as well so that the rate of growth of T 3 cultures had returned to the control level by the 5th day of T 3 exposure. Although the time frame of events is shorter in the cultured myocytes, a similar pattern of growth has been observed in intact hearts in vivo. When rats were given 20 /~g T3/100 g body weight daily for 7 days, the rate of myocardial protein synthesis increased with no change in the rate of protein breakdown [3-]. A similar increase in myocardial protein synthesis without increased protein degradation occurred in dogs given 0.5 mg T4/kg body weight daily for 7 days [2]. However, during the second week of T 4 treatment, rates of protein synthesis remained elevated but protein degradation rates also increased so that no further growth in cardiac muscle mass occurred [2]. Therefore, the pattern of early growth caused by increased protein synthesis with no change in degradation followed by a later period of increased synthesis balanced by increased degradation occurs in both cultured myocytes and intact hearts in vivo. Caution must be exercised in comparing the effects of T 3 on immature heart cells in vitro to T 3 effects on adult heart muscle in vivo. Although the contractile activity and morphology of the cultured myocytes did not change during the 5 days of T 3 exposure, the increased protein degradation observed in the cultured myocytes after 3 to 4 days of T 3 treatment may be secondary to the metabolic stress o f T 3 on myocytes grown in an artificial environment. Furthermore, cultured heart cells have much greater rates of growth and fractional protein synthesis than intact hearts of adult animals [5]. Therefore increased protein synthesis and degradation following T 3 treatment of cultured myocytes m a y not be
equivalent to similar changes following T s treatment in vivo. T 3 treatment in rats at 20 ktg per 100 g daily for 7 days affected heart and skeletal muscle protein turnover differently [3]. In heart muscle, protein synthesis and protein mass increased without any change in degradation while in skeletal muscle, both protein synthesis and degradation increased with no change in protein mass. Since T 3 treatment in vivo causes systemic changes which increase cardiac work [-9], it appeared that these secondary effects of T 3 might prevent an early direct T 3 stimulation of myocardial protein degradation. However, the fact that isolated cardiac myocytes also respond to T 3 with an early period of increased protein synthesis with no change in breakdown suggests that T 3 has a different intrinsic effect on cardiac muscle. This suggests that c~rd!ac hypertrophy in hyperthyroid animals is a primary rather than secondary effect of thyroid hormone. The low ratio of fibronectin to the myosin heavy chain fraction in cellular protein obtained from current myocyte cultures together with microscopic observations suggests that the serum-free medium was effective in preventing fibroblast overgrowth (Fig. 3). On the other hand, addition of 5% fetal bovine serum to the medium caused a marked increase in fibronectin and overgrowth of the cultures by noncontractile cells. In contrast to fetal bovine serum, 10 - s M T 3 addition did not increase the size of the fibronectin peak or alter cell morphology (Fig. 3). Therefore, the increase in the protein content of the cultures caused by T3 exposure appears to be due to stimulation of myocyte rather than fibroblast growth. To confirm that T s influences the turnover of muscle specific proteins and to show whether individual proteins are affected differently, the effect o f T 3 on the turnover of the myosin heavy chain and actin fractions was investigated. 10 -8 M T 3 increased the fractional rate of protein synthesis 24% in the myosin heavy chain fraction, 14% in the actin fraction, and 10% in total cellular protein (Table 3). Therefore, T 3 stimulated the synthesis rate of muscle specific proteins as much or more than total cellular protein. Furthermore, there was less accumulation of myosin
T 3
and M y o c y t e Protein T u r n o v e r
heavy chain protein in T 3 treated cultures when c o m p a r e d to actin, which suggests that T 3 increased the d e g r a d a t i o n of myosin heavy chain protein to a greater degree t h a n actin. E n h a n c e d myosin heavy chain t u r n o v e r in T3 treated cultures m a y be related to thyroid h o r m o n e i n d u c e d changes in the ratio of specific ventricular isomyosins that have been noted in several species [11, 17]. Since structural differences in v e n t r i c u l a r isomyosins reside in the heavy c h a i n fraction [11, 17], accelerated t u r n o v e r of the myosin heavy chain fraction would facilitate the replacem e n t of one isomyosin with another. T h e e n h a n c e d myosin heavy chain t u r n o v e r was not associated with a p p a r e n t changes in myocyte contractility. I n contrast to the rat heart, only one myosin b a n d was found in the chicken heart by native gel electrophoresis a n d it was not influenced by thyroid status [4]. Yet T 3 ,
905
treatment m a y change the myosin molecule without i n f l u e n c i n g the m i g r a t i o n of the myosin b a n d as indicated by the guinea pig heart in which thyroid status influenced myosin A T P a s e activity [7] a n d i m m u n o l o g i c reactivity [4] without altering the electrophoretic mobility of myosin [4]. Nevertheless, the e n h a n c e d myosin heavy chain t u r n o v e r observed in T 3 treated myocytes m a y not indicate a T a - i n d u c e d change in isomyosin content.
Acknowledgements This work was supported by Veterans A d m i n istration research funds, project no. 1308 002, by the N a t i o n a l Institute of Health, g r a n t No. 1-RO 1-AM20718-05, a n d by a g r a n t from the Arkansas C h a p t e r of the A m e r i c a n H e a r t Association. W e acknowledge the able technical assistance ofMax~y E. Lynch.
References l BALLARD,F.J. Regulation of protein accumulation ill cultured cells. BiochemJ 208, 275-287 (1982). 2 BONNIN,C. M., SPARROW,M. P., TAYLOR,R. R. Increased protein synthesis and degradation in the dog heart during thyroxine administration.J Mol Cell Cardiol 15, 245-250 (1983). 3 CARTER,W.j., BENJAMIN,W. S., FAAS,F. H. Effect of hyperthyroidismon protein turnover in skeletal and cardiac muscle as measured by [I*C] tyrosine infusion.BiochemJ 204, 69~74 (1982). 4 CLARK,W. A., CHIZZONITE,R. A., EVERETT,A. W., RA~INOWITZ,M., ZAK, R. Species correlations between cardiac isomyosins.A comparison of electrophoretic and immunologicalproperties.J Biol Chem 257, 5449-5454 (1982). 5 CLARK,W. A., ZAK, R. Assessment of fractional rates of protein synthesis in cardiac muscle cultures after equilibrium labeling.J Biol Chem 256, 4863-4870 (1981). 6 GASPARD,J. K., KLITGAARD,H. M., WONDERaEM,R. Somatomedin and thyroid hormones in the developing chick embryo. Proc Soc Exp Biol Med 166, 24-27 (1981) 7 GOODKIND,M. J., DAMBACn,G. E., TnVRUM,P. T., LucnI, R.J. Effect of thyroxine on ventricular myocardial contractility and ATPase activity in guinea pigs. AmJ Physio1226, 66-72 (1974). 8 HARTRE~;,E. F. A modification of the Lowry method that gives a linear photometric response. Anal Biochem 48, 422-427 (1972). 9 Hown'T, G., ROWLANDS,D. J., Lt~uNo, D. Y. T. et al. Myocardial contractility and effects of beta-adrenergic blockade in hypothyroidismand hyperthyroidism.Clin Sei 34, 485-495 (1968). 10 MARTIN,A. F., PRIOR,G., ZAK,R. Determination of specific radioactivity of amino acids in proteins directly on polyacrylamidegels: an application to L-leucine.Anal Biochem72, 577-585 (1976). 11 MORKIN,E., FLINK, I. L., GOLDMAN,S. Biochemical and physiologic effects of thyroid hormone on cardiac performance. Prog Cardiovasc Dis 25, 435-464 (1983). 12 SCHWARTZ,H., GORDON,A. Effect of triiodothyronine on chick embryo heart cells maintained in tissue culture. IsraelJ Med Sci 11,877 883 (1975). i3 SEGAL,J., GORDON,A. The effects of actinomycin D, puromycin, cycloheximide, and hydroxyurea on 3',5,3triiodo-L-thyronine stimulated 2-deoxy-D-glucoseuptake in chick embryo heart cells in vitro. Endocrinology101, 150-156 (1977). 14 SEGALJ., GORDON,A. The effect of 3',5,3-triiodo-L-thyronine on the kinetic parameters of sugar transport in cultured chick embryo heart cells. Endocrinology101, 1468-1474 (1977). 15 SEOAL,J., SCHWARTZ,H., GORDON,A. The effect of triiodothyronine on 2-deoxy-D-[1-3H] glucose uptake in cultured chick embryo heart cells. Endocrinology101, 143-149 (1977). 16 WEBER,K., OSBORN,M. The reliability of molecular weight determinations by dodecyl sulfate~-polyacrylamide gel electrophoresis.J Biol Chem 244, 4406-4412 (1969). 17 ZAK,R., CHIZZONITE,R. A., EVERETT,A. W., CLARI~,W. A. Study of ventricular isomyosinsduring normal and thyroid hormone induced cardiac growth.J Mol Cell Cardiol 14 [Suppl 3], 111-117 (1982).