Effects of Hypophysectomy, Growth Hormone, and Thyroxine on Protein Turnover in Heart*

heart content of and levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in uifro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, content and rates of protein synthesis were equal to or greater than those found in normal When plasma of acids,’ fatty acids, and and of and ventricular pressure in normal and were simulated during hearts rates of protein synthesis but rates of

From the Department of Physiology, College of Medicine, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 SUMMARY Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in uifro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids,' glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation.
Cathepsin D activity in heart homogenates ( + Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.
Hypophysectomy results in atrophy of the heart as compared to normal rats of the same body weight (1,2). During atrophy of skeletal muscle, protein synthesis was inhibited while degradation was accelerated, suggesting coordinated control of these pathways (3). On the other hand, Millward (4) reported that growth of skeletal muscle in fasted-refed rats was accompanied by faster rates of both protein synthesis and degradation. Treatment of hypophysectomized rats with either growth hormone or thyroxine stimulated growth of the heart (1,2). Stimulation of growth of skeletal muscle by growth hormone was reported to accelerate protein synthesis but to have no effect on degradation Ribosome-catalyzed reactions appear to limit the synthesis of myocardial proteins (6)(7)(8)(9). Insulin (9), availability of amino acids (lo), supply of non-carbohydrate substrates (1 l), and an increase in heart work (12) stimulated peptide-chain initiation in isolated hearts. After hypophysectomy, plasma levels of insulin, thyroxine, growth hormone, glucose, and fatty acids were reduced and the rate of ventricular pressure development was lower (13, 14). When growth hormone was added to the perfusate of isolated hearts of hypophysectomized rats, an increase in incorporation of labeled amino acids into protein occurred (15).
Protein degradation in heart muscle also appears to be under hormonal control (16). In isolated hearts, insulin inhibited degradation and increased latency of lysosomal enzymes. These changes were consistent with a model of degradation (17, 18) that involves nondifferentiated engulfment and release of cellular constituents by lysosomes. Proteins may be inactivated and denatured within the organelle. Recently, Wildenthal and Mueller (19) reported that regression of cardiac hypertrophy following cessation of thyroxine administration to thyrotoxic rats was accompanied by a 40% increase in the activity of cathepsin D. In other studies, activities of acid hydrolases increased during the period of atrophy following muscular denervation (20).
The present experiments were designed to assess the contribution of changes in the rates of protein synthesis and degradation to atrophy of the heart in hypophysectomized animals.

Heart
Perfusion--Female Sprague-Dawley rats (150 to 300 g), normal and hypophysectomized, were obtained from the Charles River Breeding Laboratory.
Rats were killed 5 to 35 days after hypophysectomy.
Hearts were rapidly excised, dropped into a beaker of 0.15 M NaCl (2"). and nerfused bv a modified Langendorff technique (21). A preliminary perfusi& was carried out for 10 min using Krebs-Henseleit bicarbonate buffer, gassed with 95y0 02-5y0 CO* and containing glucose (15 mM) and amino acids at the level to be present during the subsequent period of recirculation. This buffer passed through the heart a single time and was discarded. Recirculation of a measured volume of buffer containing [i4C]phenylalanine, other nonradioactive amino acids at 1 or 5 times normal plasma levels, and glucose .or albumin-bound (4%) palmitate (11) followed the preliminary perfusion. These amino acid levels were reoorted earlier (10). The first 10 ml of radioactive buffer were washed through t'he'heart and discarded to reduce dilution of phenylalanine specific activity. Recirculation heart weight fell by 25%; after 2 weeks, heart size stabilized at 65y0 of the preoperative value (1,2,27). Treatment of hypophysectomized rats with growth hormone, thyroxine, and a combination of growth hormone and thyroxine increased heart weight by 15, 29, and 45TC,, respectively (27).
Treatment of normal animals with a combination of these hormones had no effect on heart weight. Since protein synthesis in heart muscle is stimulated by increasing levels of insulin and fatty acids (9, II), these levels were measured in the serum of hypophysectomized rats. Serum levels of free fatty acid were 0.517 & 0.017 mM (11 observations), 0.527 =t 0.012 mM, and 0.439 + 0.009 mM (13 observations) 5, 15, or 35 days following hypophysectomy, respectively. These levels in unoperated paired controls averaged 0.562 A= 0.014 (23 observations) and did not change significantly during this time period. Insulin levels were 21 h 3.3 microunits/ml (4 observations) and 18 =t 3 microunits/ml (4 observations) at 15 and 35 days after operation, as compared to 44 + 2.3 microunits/ ml (4 observations) in unoperated controls.
Levels of ribosomal subunits and polysomes reflect relative rates of peptide-chain initiation and elongation (9). Hearts of rats that were hypophysectomized 5 to 15 days before death con tained increased levels of ribosomal subunits and reduced levels of polysomes (Table I). These findings were consistent with a relatively greater restraint on initiation than elongation of chains during the period of atrophy. Thirty days after hypophysectomy, when a smaller but stable heart size was achieved, subunit and polysome levels were not significantly different from normal. Treatment of hypophysectomized rats with growth hormone and thyroxine reduced levels of ribosomal subunits and increased levels of polysomes, suggesting that the hormones were able to restore the normal relationship between rates of initiation and elongation of peptide chains. In other experiments, treatment of normal rats with growth hormone and thyroxine had no effect on levels of polysomes and subunits. During the rapid phase of cardiac atrophy (5 days, postoperative) RNA levels were reduced by 12$& (Table I). When a stable heart size was achieved, RNA levels in hearts of hormone-deficient rats returned to normal. Injections of growth hormone increased RNA to normal levels. Thyroxine, or a combination of both hormones, increased RNA content in hearts of hypophysectomized rats by approximately 20%. Injection of both hormones raised RNA content of normal hearts by 10%.
Measurement of Protein. Sylzthesis-Earlier studies (27) showed that the rate of phenylalanine incorporation into protein was the same in hearts of normal and hypophysectomized rats during perfusion for 1 hour with buffer containing 0.08 mM phenylalanine and 15 mM glucose. Under these conditions, levels of polysomes fell and ribosomal subunits increased, indicating that a block in peptide-chain initiation was present in both groups of hearts. In the present experiments, hearts were perfused (a) with buffer containing 5 times normal plasma levels of amino acids and albumin-bound palmitate to give maximal rates of synthesis (11) ; or (b) with buffer containing levels of amino acids, fatty acids, glucose, and insulin that approximated those found in the serum of normal and hormone-deficient animals. In the latter groups, rates of ventricular pressure development and heart rate In experimental series I, growth hormone (GH), thyroxine (Td), or both hormones (GH,T,) were injected for 7 days prior to death, as indicated. Hearts were perfused for 1 hour with buffers containing palmitate (1.5 mM)-albumin (4q7'), 5 times normal plasma levels of amino acids (lo), and ["Clphenylalanine at a specific activity of 595,000 dpm/rmol. When hearts were perfused under these conditions, the lower rate of protein synthesis in hearts of hypophysectomized rats was not associated with low intracellular levels of free amino acids (27). In the case of each amino acid, the level in these hearts was equal to or greater than those found in unperfused normal hearts. However, levels of glutamic acid and serine were reduced by 28 and 42%, respectively, as compared to normal hearts perfused in the presence of palmitate and 5 times normal plasma levels of amino acids (27 also were adjusted to more closely simulate normal and hypophysectomized conditions. When hearts from normal rats were perfused with buffer containing palmitate and 5 times normal plasma levels of amino acids (Table II), RNA content of sucrose gradient peaks representing the large and small ribosomal subunits were 0.215 f 0.015 and 0.101 f 0.020 mg of RNA/3 mg of RNA in the heart homogenate, respectively (3 observations).
Hearts of hypophysectomized rats, perfused under these conditions, contained 0.181 =t 0.030 and 0.079 + 0.020 mg of RNA in these gradient fractions (3 observations). None of these values were significantly different from those found in normal unperfused hearts. Rates of protein synthesis were lower in hearts of hypophysectomized rats (5 to 15 days after operation) than in hearts of normal rats (Table  II, Series I). After a longer period (24 to 30 days), protein synthesis occurred at the same rate in both groups of hearts.
The reduced rate of protein synthesis in hearts from hypophysectomized rats (5 to 15 days after operation) could have been due to a delay in reaggregation of ribosomes at the beginning of the perfusion period. A delay of 30 min occurred before palmitate fully reaggregated ribosomal subunits in perfused hearts of normal rats (II). When hearts were perfused for 2 hours in the presence of palmitate and 5 times normal plasma levels of amino acids, rates of protein synthesis during the second hour were lower in hearts from hypophysectomized rats (Table II, Series II) even though the ribosomes were reaggregated.
Reaggregation of ribosomal subunits in hearts of hypophysectomized rats did not appear to depend on synthesis of mRNA (Fig. 1). After 1 hour of perfusion with buffer containing actinomycin D, levels of ribosomal subunits were lower than in unperfused hearts of hypophysectomized rats, but similar to levels found in normal hearts.
Treatment of hypophysectomized rats with growth hormone or thyroxine (or both) increased the rate of protein synthesis (Table II

Degradation and Lysosomal Enzyme
Activities-In the first series of experiments (Table IV), net release of phenylalanine and protein degradation were measured in hearts perfused with buffer containing glucose and amino acids. Net release, reflecting the balance between rates of protein synthesis and degradation, occurred in both groups of hearts, but was lower in hearts from hypophysectomized animals (5 to 35 days, postoperative).
Protein degradation was somewhat lower 15 days after hypophysectomy.
In Series II, net release of phenylalanine was not detected in hearts of normal or hypophysectomized animals perfused in the presence of insulin. The hormone reduced protein degradation about 507,. Rates of degradation in hearts of hypophysectomized rats were the same as in normal hearts.
During the period of rapid atrophy following hypophysectomy (5 to 8 days, postoperative), total activity of cathepsin D increased while the fraction assayable in the absence of Triton was the same as in normal hearts (Table V). The fraction of cathepsin D activity recovered in the lo4 x g pellet was increased somewhat 5 days after hypophysectomy.
On the other hand, total activity of P-acetylglucosaminidase was lower in hearts of hypophysectomized rats (5 days postoperative) but a higher fraction of total activity was recovered in the lo4 x g pellet. The fraction of activity assayable in the absence of Triton was the same as in normal hearts after 5 days but increased somewhat 8 and 14 days postoperatively.
These changes in the total activities of cathepsin D and P-acetylglucosaminidase were similar to those reported by Wildenthal and Mueller (19). DISCUSSION When normal and hypophysectomized conditions were simulated in vitro as had been done with protein synthesis, neither net release of phcnylalanine nor the rate of protein degradation were significantly different (Series III). However, the difference between these rates, which gave an approximation of the rate of protein synthesis, was greater under normal conditions (0.12 + 0.01 pmol of phenylalanine/g~ hour) than under hypophysectomized conditions (0.08 =t 0.01). A more direct assessment of degradation rates was obtained by measuring net release of phenylalanine in the presence of cycloheximide.

Release was
Plasma levels of insulin, fatty acids, and amino acids, and tissue levels of high energy phosphates in normal animals are sufficient to accelerate peptide-chain initiation in heart muscle and to shift the restraint on protein synthesis to reactions involved in elongation and termination of chains (9, 11). These findings suggested that protein synthesis was limited by the quantity of ribosomes available to take part, in formation of peptide bonds.
During cardiac atrophy in hypophysectomized rats, two changes may have contributed to a decreased rate of protein synthesis. The first is a reduction in the total RNA'per g of heart, reflecting a reduction in the number of ribosomes, and the second is an imbalance between rates of initiation and elongation of chains. In the latter case, a reduction in polysomes and an increase in ribosomal subunits is consistent with inhibition of chain initiation; alternatively, these changes could result from accelerated rates of chain elongation and termination.
This possibility  would be contrary to that expected during cardiac atrophy. When plasma levels of insulin, glucose, fatty acids, and amino acids, and the mechanical performance of hearts of hypophysectomized rats was simulated in vitro, levels of ribosomal subunits fell. Thus, the imbalance between rates of initiation and elongation that was found in viva could not be reproduced in vitro. Factors accounting for this imbalance in vivo are unknown. Reversal of the changes in levels of RNA, ribosomal subunits, and polysomes followed treatment of hypophysectomized rats with growth hormone and thyroxine or elapse of sufficient time for the heart to reach a stable smaller size. Hormonal treatment resulted in rapid growth of the heart in association with growth of the rat, while in the hormone-deficient animal no such growth occurred. These results raise the question of whether the hormones had a direct effect on turnover of RNA and protein or whether the hormones exerted their effect by changing the contractility of the heart and the work load imposed upon it. In earlier experiments, hypophysectomy was associated with impaired cardiac contractility and decreased activity of myosin ATPase (14). These changes were reversed by treatment of the rats with thyroxine. Impaired incorporation of precursors into myocardial RNA of hypophysectomized rats has been reported (33) Cardiac atrophy indicated that the rate of protein degradation exceeded that of synthesis. In previous experiments, rates of protein synthesis and degradation in hearts perfused with buffer containing added insulin indicated that turnover amounted to about 10% of heart protein per day (16). Measurements in intact rats suggested that approximately 13.6% of heart protein turned over each day (29). In the present experiments, approximately 25% of cardiac mass was lost in the first 8 days after hypophysectomy, suggesting a net degradation rate of about 3% per day. In hormone-deficient hearts, the fall in levels of ribosomes could be expected to reduce the rate of protein synthesis about 15%. This factor would suggest that 11.6 rather than 13.6% of heart protein would be synthetized each day, and would

Animal
Cathepsin D activity (2 X lO+.cpm, g.30 min) Normal leave 1.0% per day to be accounted for either by a restraint on peptide-chain initiation or an accelerated rate of degradation. Measurements of protein degradation during in vitro perfusion of normal and hypophysectomized rats were undertaken in an attempt to determine whether the rate was modified in hormone-deficient hearts. Perfusion with buffer containing amino acids and glucose resulted in phenylalanine release, but the rate was lower in hormone-deficient hearts. Degradation was either unchanged or reduced in hearts of hypophysectomized as compared to normal rats. When insulin was added to the perfusate, net release of phenylalanine was zero in both groups of hearts and degradation was reduced by 40 to 50%. When normal and hypophysectomized conditions were simulated in vif~o, net release and protein degradation were the same under both conditions of perfusion. Measurement of degradation by this method underestimated the rate by 357, due to reincorporation of non radioactive phenylalanine prior to mixing with the total pool of [%]phenylalanine (16). When protein degradation was measured in hearts in which protein synthesis was inhibited with cycloheximide, the rates were the same under simulated normal and hypophysectomized conditions. These rates also were underestimated about 20% due to inhibition of proteolysis by the drug (16,30,31). In order to obtain an additional assessment of the rate of degradation, phenylalanine release (Table IV) and protein synthesis (Table III) were summed. Under simulated normal and hypophysectomized conditions, rates of proteolysis were 0.26 and 0.24 pmol of phenylalanine/g of heart. hour, respectively. These measurements, under a variety of in vitro conditions, indicated that protein degradation was not increased in hearts of hypophysectomized rats undergoing atrophy. However, rates of degradation measured in vitro may not faithfully reflect the in vivo rate, as indicated by net release of phenylalanine under simulated normal conditions. A model of protein degradation (17, 18) involving lysosomes was suggested to account for protein degradation in heart muscle (16). Enzymatic inactivation and denaturation within the organelles would depend upon the susceptibility of individual proteins to proteolysis. During atrophy of hearts of hypophysectomized rats, the total activity of cathepsin D, as assayed in the whole homogenate in the presence of Triton, increased about 19%. The percentage of total activity assayable without Triton was unchanged while that sedimentable at lo4 x g was higher.
Total activity of P-acetylglucosaminidase was not increased. Thus, changes in total activity of cathepsin D did not reflect rates of protein degradation.
When activities of lysosomal enzymes were expressed per heart rather than per g of heart, total activity of cathepsin D was unchanged (normal, 318,000 & 2,500; hypophysectomized, 323,500 + 7,500 cpm/heart. 30 min) while activity of P-acetylglucosaminidase was decreased 5 days after hypophysectomy (normal, 8.6 & 0.03; hypophysectomized, 7.4 =t 0.07 pmol/ heart.30 min). These data indicated that the higher total activity of cathepsin D need not be attributed to increased synthesis of the enzyme but could have resulted from slower degradation of cathepsin D than of whole heart protein. On the other hand, @-acetylglucosaminidase activity decreased in proportion to heart size. As an alternative to differing rates of enzyme degradation, these results could reflect heterogenity of lysosomes within