Direct anabolic effects of thyroid hormone on isolated mouse heart

The direct effects of L-and D-triiodothyronine (T3) on cardiac protein metabolism were investigated using fetal mouse hearts in organ culture. This model allowed the production of "thyrotoxicosis" in isolated hearts in vitro in the absence of the usual systemic metabolic and hemodynamic effects of thyroid hormones. Hearts were studied during the first 24 h of T3 exposure in culture, before changes in beating rate due to T3 occurred. Phenylalanine release was decreased by 26 +/- 2.3% (P less than 0.001) by the optimal concentrations of T3 (10(-7) to 10(-6) M). Changes were similar in the presence or absence of insulin. D-T3 was also anabolic, decreasing phenylalanine release by 24 +/- 2.5% (P less than 0.001) at concentrations of 10(-6) to 10(-5) M. The L-isomer increased protein synthesis by 23 +/- 6.8% (P less than 0.05) and decreased protein degradation, as measured by phenylalanine release in the presence of cycloheximide, by 5 +/- 1.6% (P less than 0.01). The D-isomer also increased protein synthesis but had no measurable effect on protein degradation. We conclude that thyroid hormones can exert direct anabolic effects on heart in the absence of systemic hemodynamic and metabolic changes. These effects are mediated primarily through an acceleration of the rate of protein synthesis; in the case of L-T3, a small inhibition of proteolysis may also occur.

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Regulation by thyroid status of c-myc, c-fos and H-ras mRNAs in the rat myocardium

Journal of Endocrinology ◽

10.1677/joe.0.1300239 ◽

1991 ◽

Vol 130 (2) ◽

pp. 239-244 ◽

Cited By ~ 5

Author(s):

N. K. Green ◽

M. D. Gammage ◽

J. A. Franklyn ◽

M. C. Sheppard

Keyword(s):

Protein Synthesis ◽

Thyroid Hormones ◽

Contractile Protein ◽

Rat Myocardium ◽

Direct Effects ◽

Thyroid Status ◽

Critical Signal ◽

Ras Genes ◽

Intracellular Signals ◽

Oncogene Products

ABSTRACT Effects of thyroid status on expression of a variety of myocardial genes, such as those encoding contractile proteins, have been reported, as well as interactions between thyroid hormones and developmental and haemodynamic regulation of contractile protein synthesis. In addition, it is clear that developmental and haemodynamic factors regulate expression of specific proto-oncogenes, including c-myc, c-fos and H-ras, in the myocardium but the effect of thyroid status on such proto-oncogene products, which are proposed to play a critical signal-transducing role in the heart, has been previously unexplored. In order to determine whether changes in thyroid status are associated with changes in expression of these putative intracellular signals, we examined the effect of hypothyroidism and tri-iodothyronine (T3) treatment on myocardial levels of c-myc, c-fos and H-ras mRNAs in the rat. The induction of hypothyroidism was associated with a marked increase in myocardial c-myc, c-fos and H-ras mRNAs, changes reversed by 72 h of T3 replacement. Administration of T3 to euthyroid rats had no significant effect on myocardial c-myc or c-fos mRNAs, but inhibition of H-ras mRNA by T3 was evident. These observations demonstrating influences of thyroid status on expression of specific proto-oncogenes suggest that thyroid hormones, as well as exerting direct effects on expression of functionally important myocardial genes, also interact with the cellular transduction pathways mediated by the products of the c-myc, c-fos and H-ras genes. Journal of Endocrinology (1991) 130, 239–244

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Effects of insulin in vitro on protein turnover in rat epitrochlearis muscle

Biochemical Journal ◽

10.1042/bj2100323 ◽

1983 ◽

Vol 210 (2) ◽

pp. 323-330 ◽

Cited By ~ 41

Author(s):

W S Stirewalt ◽

R B Low

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Nitrogen Balance ◽

Protein Metabolism ◽

Serum Insulin ◽

Specific Radioactivity ◽

Physiological Concentration ◽

Rat Serum ◽

The Third

Rates of protein synthesis and degradation were measured in the isolated rat epitrochlearis muscle by radiotracer techniques, by using the specific radioactivity of tRNA-bound amino acid as precursor for protein synthesis. The tissue maintained linear rates of protein synthesis for 3 h of incubation in the presence of amino acids and glucose and in the absence of insulin. Under these conditions, however, the muscles were in negative nitrogen balance, with rates of protein degradation exceeding rates of protein synthesis. Under steady-state conditions of labelling, the specific radioactivities of tRNA-bound leucine, phenylalanine and valine were significantly less than their respective values in the incubation medium, at concentrations in the medium varying from 1 to 10 times those in normal rat serum. Insulin caused a dose- and time-dependent increase in tRNA-based protein synthesis rates, more than doubling rates at 5 and 50 ng of insulin/ml. At the lower, physiological, concentration of insulin, the stimulation of protein synthesis was not observed until the third hour of incubation with the hormone, whereas the rate of protein synthesis at the higher concentration was elevated during the second hour. There were no delays in the stimulation by insulin of glucose conversion into glycogen. The delayed stimulatory effects of insulin on the rate of protein synthesis brought the tissue to a nitrogen balance near zero. The presence of the hormone also prevented the increase in the rate of protein degradation seen in the third hour of incubation in the absence of the hormone. These studies demonstrate the viability of the incubated rat epitrochlearis muscle with respect to protein metabolism and sensitivity to the protein anabolic effects of physiological concentrations of insulin, and indicate that the preparation is a suitable experimental model for the study of the control of protein metabolism in fast-twitch skeletal muscle.

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Comparison of protein synthesis and degradation in incubated and perfused muscle

Biochemical Journal ◽

10.1042/bj2120649 ◽

1983 ◽

Vol 212 (3) ◽

pp. 649-653 ◽

Cited By ~ 35

Author(s):

A S Clark ◽

W E Mitch

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Insulin Treatment ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Muscle Protein Degradation ◽

Protein Synthesis And Degradation ◽

Synthesis And Degradation

Rates of muscle protein synthesis and degradation measured in the perfused hindquarter were compared with those in incubated epitrochlearis muscles. With fed or starved mature rats, results without insulin treatment were identical. With insulin treatment, protein synthesis in perfused hindquarters was greater, though protein degradation was the same. Thus rates of muscle protein degradation estimated by these two methods in vitro correspond closely.

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Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

Circulation Research ◽

10.1161/res.109.suppl_1.ap180 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Kedryn K Baskin ◽

Rebecca Salazar ◽

Wenhao Chen ◽

Heinrich Taegtmeyer

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Fold Increase ◽

Ubiquitin Proteasome System ◽

Initiation Factor ◽

Skeletal Muscle Atrophy ◽

Heart Weight ◽

Cross Sectional

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

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The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation

Biochemical Journal ◽

10.1042/bj1880247 ◽

1980 ◽

Vol 188 (1) ◽

pp. 247-254 ◽

Cited By ~ 15

Author(s):

M J Seider ◽

R Kapp ◽

C P Chen ◽

F W Booth

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Extensor Digitorum Longus ◽

High Energy ◽

Extensor Digitorum ◽

Muscle Fibres ◽

High Energy Phosphates ◽

Protein Synthesis And Degradation ◽

Synthesis And Degradation

Rates of protein synthesis were significantly lower in the cut soleus and extensor digitorum longus muscles than in their uncut counterparts. Rates of protein degradation were significantly higher in cut soleus muscles, but not in cut extensor digitorum longus muscles as compared with their uncut controls. Concentrations of ATP and phosphocreatine were significantly lower in cut soleus and extensor digitorum longus muscles after incubation in vitro in contrast with respective control uncut muscles. These data indicate that cutting of muscle fibres alters rates of protein synthesis and degradation, in addition to altering concentrations of high-energy phosphates. Since these findings stressed the importance of using intact muscles to study protein metabolism, additional studies were made on intact muscles in vitro. Stretched soleus muscles had higher concentrations of high-energy phosphates at the end of an incubation period than did unstretched muscles. However, the length of the soleus, extensor digitorum longus and diaphragm muscles during incubation did not affect rates of protein degradation.

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Rumen protein degradation and biosynthesis

British Journal Of Nutrition ◽

10.1079/bjn19830128 ◽

1983 ◽

Vol 50 (3) ◽

pp. 569-582 ◽

Cited By ~ 68

Author(s):

L. Raab ◽

B. Cafantaris ◽

T. Jilg ◽

K. H. Menke

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Rumen Fluid ◽

Ammonia Concentration ◽

Gas Production ◽

Total N ◽

Fluid Medium ◽

The Difference ◽

Fermentable Carbohydrates

1. A method is described for the determination of protein degradation based on measurements of ammonia concentration and gas production (Menke et al. 1979) when a feedingstuff was incubated with rumen fluid in vitro.2. NH3 liberated during incubation is in part used for microbial protein synthesis. Production of carbon dioxide and methane can be regarded as a measure of energy available for protein synthesis. The ratio, gas production: incorporation of NH3-nitrogen was estimated by addition of starch to the substrate. The response in gas production was linear in the range 0–200 mg starch, when starch was added to 0–200mg feedingstuff dry matter and 30 ml rumen fluid-medium mixture.3. Linear regression between NH3-N concentration (y, mg) and gas production (x, ml) yielded an intercept (b0) representing that amountof NH3-N which would be released when no fermentable carbohydrates were available and consequently no bacterial protein synthesis took place.4. The difference between this intercept b0 and NH3-N content in the blank (rumen fluid without substrate added) indicated the amount of NH3 liberated from protein and other N-containing compounds of the feedingstuff incubated. In vitro-degradable N (IVDN) was calculated as a proportion of total N by the equation:

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Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats

Biochemical Journal ◽

10.1042/bj2220395 ◽

1984 ◽

Vol 222 (2) ◽

pp. 395-400 ◽

Cited By ~ 34

Author(s):

V R Preedy ◽

D M Smith ◽

N F Kearney ◽

P H Sugden

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Protein Turnover ◽

Synthesis Rate ◽

Protein Synthesis Rate ◽

Ventricular Muscle ◽

Degradation Rates ◽

Perfused Hearts

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.

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Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a294 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Kedryn K Baskin ◽

Rebecca Salazar ◽

Wenhao Chen ◽

Heinrich Taegtmeyer

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Skeletal Muscle Atrophy ◽

Heart Weight ◽

Cross Sectional ◽

Mechanical Unloading ◽

Ubiquitin Proteasome ◽

Specific Proteins

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

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