Turnover of muscle protein in the fowl (Gallus domesticus). Rates of protein synthesis in fast and slow skeletal, cardiac and smooth muscle of the adult fowl.

Rates of protein synthesis in skeletal, cardiac and smooth muscle of fully grown fowl (Gallus domesticus) were determined in vivo by means of the constant infusion method using [14C]proline. In the anterior latissimus dorsi muscle, containing predominantly slow fibres, the average synthesis rate of non-collagen muscle proteins was 17.0 +/- 3.1% per day, a value higher than that obtained for cardiac muscle (13.8 +/- 1.3% per day) and for smooth muscle of the gizzard (12.0 +/- 1.9% per day). In the posterior latissimus dorsi muscle, containing predominantly fast fibres, synthesis rates were much lower (6.9 +/- 1.8% per day). In each case these average rates for the non-collagen protein were similar to the average rate for the sarcoplasmic and myofibrillar protein fractions. The RNA concentration of these four muscles showed that relative rates of protein synthesis were determined mainly by the relative RNA concentrations. The rate of protein synthesis per unit of DNA (the DNA activity) was similar in the two skeletal muscles, but somewhat lower in cardiac muscle and gizzard, possibly reflecting the larger proportion of less active cell types in these two muscles. These quantitative aspects of protein turnover in the two skeletal muscles are discussed in terms of the determination of ultimate size of the DNA unit, and in relation to muscle ultrastructure.     

Turnover of muscle protein in the fowl. Changes in rates of protein synthesis and breakdown during hypertrophy of the anterior and posterior latissimus dorsi muscles.

Measurements were made of the growth and of the changes in rates of protein turnover in the anterior latissimus dorsi muscle of the adult fowl in response to the attachment of a weight to one wing. Over 58 days there was a 140% increase in the protein content with similar increases in the RNA and DNA contents. The fractional rate of protein synthesis, measured by the continuous-infusion technique using [14C]proline, increased markedly during hypertrophy. This increase was mediated initially (after 1 day) by an increase in the RNA activity but at all other times reflected the higher RNA content. The rate of protein degradation, calculated from the difference between the synthesis and growth rates, appeared to increase and remain elevated for at least 4 weeks. At no time was there any suggestion of a fall in the rate of degradation. The following events are discussed as central to the changes that occur during skeletal-muscle hypertrophy. 1. Nuclear proliferation is necessary to maintain the characteristic synthesis rate because of the inability of existing nuclei to 'manage' increased protein synthesis for more than a limited period. 2. The increased protein breakdown during hypertrophy is consistent with the known over-production of a new muscle fibres and may indicate some 'wastage' during the growth. Such wastage may also be associated with myofibrillar proliferation. 3. Muscle stretch must be recognized as the major activator of growth and as such can be compared with the 'pleiotypic activators' that have been described for cells in culture.     

The effect of intermittent changes in tension on protein and collagen synthesis in isolated rabbit muscles.

Isolated intact rabbit muscles were incubated in a medium containing radioactive proline. The rates of synthesis of collagen and total muscle protein after incubation with a constant tension or intermittent mechanical stretching were compared with the rates in vivo. Muscles incubated under a constant tension synthesized protein at 22% of the rate observed in vivo; intermittent mechanical stretching resulted in an increase of 73% in the rate of protein synthesis, to 38% of that found in vivo. Collagen synthesis was affected in the same way as total protein synthesis by both types of incubation, therefore the relative rates of collagen and total protein synthesis were unchanged. ATP concentration in the isolate muscles and the uptake of glucose from the medium were increased by intermittent mechanical stretching. Incubating the muscles with a gas phase containing 5% O2 decreased the rate of protein synthesis, abolished the effect of intermittent mechanical stretching, lowered the concentration of ATP and increased the lactate concentration. The rate of protein synthesis in muscles maintained with a constant or intermittently applied tension was not affected by a previous period of incubation with the other type of stimulus.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Effect of heart failure on the regulation of skeletal muscle protein synthesis,breakdown, and apoptosis

Heart failure is often characterized by skeletal muscle atrophy. The mechanisms underlying muscle wasting, however, are not fully understood. We studied 30 Dahl salt-sensitive rats (10 male, 20 female) fed either a high-salt (HS; n = 15) or a low-salt (LS; n = 15) diet. This strain develops cardiac hypertrophy and failure when fed a HS diet. LS controls were matched to HS rats for gender and duration of diet. Body mass, food intake, and muscle mass and composition were measured. Skeletal muscle protein synthesis was measured by isotope dilution. An additional group of 27 rats (HS, n = 16; LS; n = 11) were assessed for expression of genes regulating protein breakdown and apoptosis. Gastrocnemius and plantaris muscles weighed less (16 and 22%, respectively) in HS than in LS rats (P < 0.01). No differences in soleus or tibialis anterior weights were found. Differences in muscle mass were abolished after data were expressed relative to body size, because HS rats tended (P = 0.094) to weigh less. Lower body mass in HS rats was related to a 16% reduction (P < 0.01) in food intake. No differences in muscle protein or DNA content, the protein-to-DNA ratio, or muscle protein synthesis were found. Finally, no differences in skeletal muscle gene expression were found to suggest increased protein breakdown or apoptosis in HS rats. Our results suggest that muscle wasting in this model of heart failure is not associated with alterations in skeletal muscle metabolism. Instead, muscle atrophy was related to reduced body weight secondary to decreased food intake. These findings argue against the notion that heart failure is characterized by a skeletal muscle myopathy that predisposes to atrophy.     

mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Longitudinal gradients of collagen and elastin gene expression in the porcine aorta

The physical and chemical properties of the mammalian aorta are known to vary as a function of distance from the heart. These properties are highly dependent collagen and elastic fibers. In order to evaluate the mechanisms which regulate the accumulation of these two connective tissue proteins, gene expression was evaluated at both the biosynthetic and messenger RNA levels. Short-term (3 h) explant cultures of the medial portion of four segments of the descending aorta in newborn pigs were incubated in the presence of [3H] proline. Collagen production was quantified by collagenase digestion and elastin production was determined by immunoprecipitation. Between the conus arteriosus and the bifurcation of the iliac arteries, relative collagen synthesis increased 2-fold (from 5.8 to 12.0% of total protein synthesis), while relative elastin synthesis declined 10-fold (from 16.4 to 1.6% of total protein synthesis). Similarly, collagen production increased more than 7-fold (from 6.7 to 49.8 X 10(3) molecules/cell/h) while elastin production was reduced more than 3-fold (from 71.8 to 21.0 X 10(3) molecules/cell/h) along this developmental gradient. Elastin synthesis appeared to be controlled to a significant extent by the availability of elastin mRNA, since both cell-free translation and molecular hybridization to a cloned elastin gene probe showed gradients of elastin gene expression. Similarly, collagen synthesis was apparently regulated, at least in part, by an inverse gradient of collagen mRNA, as measured with a cloned cDNA for the pro-alpha 1(I) collagen gene. Marked changes in the amount of non-elastin protein synthesis accompanied differentiation and accounted for larger changes in relative synthesis. These results suggest that the phenotype of the cells of the porcine artery wall is distinct in different regions of this organ at this developmental stage.     

Nifedipine does not impede clenbuterol-stimulated muscle hypertrophy.

The mechanism(s) responsible for beta2-adrenergic receptor-mediated skeletal muscle and cardiac hypertrophy remains undefined. This study examined whether calcium influx through L-type calcium channels contributed to the development of cardiac and skeletal muscle (plantaris; gastrocnemius; soleus) hypertrophy during an 8-day treatment with the beta2-adrenergic receptor agonist clenbuterol. Concurrent blockade of L-type calcium channels with nifedipine did not reverse the hypertrophic action of clenbuterol. Moreover, nifedipine treatment alone resulted in both cardiac and soleus muscle hypertrophy (6% and 7%, respectively), and this effect was additive to the clenbuterol-mediated hypertrophy in the heart and soleus muscles. The hypertrophic effects of nifedipine were not associated with increases in total beta-adrenergic receptor density, nor did nifedipine reverse clenbuterol-mediated beta-adrenergic receptor downregulation in either the left ventricle or soleus muscle. Both nifedipine and clenbuterol-induced hypertrophy increased total protein content of the soleus and left ventricle, with no change in protein concentration. In conclusion, our results support the hypothesis that beta2-adrenergic receptor agonist-induced muscle hypertrophy is mediated by mechanisms other than calcium influx through L-type calcium channels.     

Pre- and post-natal growth and protein turnover in smooth muscle, heart and slow- and fast-twitch skeletal muscles of the rat.

The growth of one smooth and three individual striated muscles was studied from birth to old age (105 weeks), and where possible during the later stages of foetal life also. Developmental changes in protein turnover (measured in vivo) were related to the changing patterns of growth within each muscle, and the body as a whole. Developmental growth (i.e. protein accumulation) in all muscles involved an increasing proportion of protein per unit wet weight, as well as cellular hypertrophy. The contribution of the heart towards whole-body protein and nucleic acid contents progressively decreased from 18 days of gestation to senility. In contrast, post-natal changes in both slow-twitch (soleus) and fast-twitch (tibialis anterior) skeletal muscles remained reasonably constant with respect to whole-body values. Such age-related growth in all four muscle types was accompanied by a progressive decline in both the fractional rates of protein synthesis and breakdown, the changes in synthesis being more pronounced. Age for age, the fractional rates of synthesis were highest in the oesophageal smooth muscle, similar in both cardiac and the slow-twitch muscles, and lowest in the fast-twitch tibialis muscle. Despite these differences, the developmental fall in synthetic rates was remarkably similar in all four muscles, e.g. the rates at 105 weeks were 30-35% of their values at weaning. Such developmental changes in synthesis were largely related to diminishing ribosomal capacities within each muscle. When measured under near-steady-state conditions (i.e. 105 weeks of age), the half-lives of mixed muscle proteins were 5.1, 10.4, 12.1 and 18.3 days for the smooth, cardiac, soleus and tibialis muscles respectively. Old-age atrophy was evident in the senile animals, this being more marked in each of the four muscle types than in the animal as a whole. In each muscle of the senile rats the protein content and composition per unit wet weight, and both the fractional and total rates of synthesis, were significantly lower than in the muscles of younger, mature, animals (i.e. 44 weeks). In the soleus the decreased synthesis rate appeared to be related to a further fall in the ribosomal capacity. In contrast, the changes in synthesis in the three remaining muscles correlated with significant decreases in the synthetic rate per ribosome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Collagen synthesis in explants from rat intestine

Collagen is the major structural component of the intestinal wall and its metabolism is of special interest for intestinal strength. We describe collagen synthesis in short-term (3 h) incubations of rat intestinal tissue, as measured in terms of incorporation of [3H]proline in collagenase-digestible protein and percentage relative collagen synthesis. In this time span, incorporation of [3H]proline in collagen increases linearly with time and tissue weight. Addition of unlabeled proline during incubation, in excess of the 0.1 microM [3H]proline always present, strongly increases both total protein and collagen synthesis, suggesting that proline transport is rate limiting. Further experiments have been performed in the presence of labeled proline alone and with the addition of 0.35 mM unlabeled proline. Collagen synthesis is significantly higher in colon than in ileum, comprising 0.37 and 0.21%, respectively, of total protein synthesis. Also, collagen synthesis decreases considerably with age, both in ileum and colon. The results presented here demonstrate that rat intestinal explants synthesize measurable amounts of collagen in vitro and that the system used is able to detect changes in in vivo synthetic capability such as those induced by ageing.     

Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats

Insulin-like growth factor I (IGF-I) peptidelevels have been shown to increase in overloaded skeletal muscles (G. R. Adams and F. Haddad. J. Appl.Physiol. 81: 2509-2516, 1996). In that study, theincrease in IGF-I was found to precede measurable increases in muscleprotein and was correlated with an increase in muscle DNA content. Thepresent study was undertaken to test the hypothesis that direct IGF-Iinfusion would result in an increase in muscle DNA as well as invarious measurements of muscle size. Either 0.9% saline or nonsystemicdoses of IGF-I were infused directly into a non-weight-bearing muscleof rats, the tibialis anterior (TA), via a fenestrated catheterattached to a subcutaneous miniosmotic pump. Saline infusion had noeffect on the mass, protein content, or DNA content of TA muscles.Local IGF-I infusion had no effect on body or heart weight. Theabsolute weight of the infused TA muscles was ~9% greater(P < 0.05) than that of thecontralateral TA muscles. IGF-I infusion resulted in significantincreases in the total protein and DNA content of TA muscles(P < 0.05). As a result of thesecoordinated changes, the DNA-to-protein ratio of the hypertrophiedTA was similar to that of the contralateral muscles. These resultssuggest that IGF-I may be acting to directly stimulate processes suchas protein synthesis and satellite cell proliferation, which result inskeletal muscle hypertrophy.

     

Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Subcellular responses of p53 and Id2 in fast and slow skeletal muscle in response to stretch-induced overload.

Tumor suppressor p53 and inhibitor of DNA-binding/differentiation Id2 were examined after 7 or 21 days of wing weighting in fast patagialis (PAT) and slow anterior latissimus dorsi (ALD) wing muscles of young adult and old Japanese quails. The contralateral wing served as the intra-animal control. Seven days of loading increased PAT and ALD muscle weight by 28 and 96%, respectively, in young birds. PAT and ALD muscle weight was 49 and 179% greater, respectively, than control muscles after 21 days of loading in young birds. In aged birds, no PAT or ALD hypertrophy was found after 7 days of loading; however, PAT and ALD muscle weight increased by 29 and 102%, respectively, after 21 days of loading. Id2 protein in the nuclear muscle fraction increased in both PAT and ALD muscles from young adult and old birds that were loaded for 7 days and in ALD muscles after 21 days of loading relative to contralateral control muscles. Nuclear p53 protein was greater in 7- or 21-day loaded PAT and ALD muscles relative to control muscles in both age groups. Cytosolic Id2 and p53 protein contents were not changed in loaded PAT or ALD muscles relative to control muscles at any time point. These data suggest that nuclear, but not cytosolic, Id2 and p53 are responsive to stretch-induced muscle overload. Moreover, the attenuated ability of the aged skeletal muscle to achieve hypertrophy does not appear to be explained by the subcellular changes in Id2 and p53 content with overload.     

Heat stress inhibits skeletal muscle hypertrophy

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.     

Age-related apoptotic responses to stretch-induced hypertrophy in quail slow-tonic skeletal muscle

In the present study, we examined the responses of apoptosis and apoptotic regulatory factors to muscle hypertrophy induced by stretch overload in quail slow-tonic muscles. The wings from one side of young and aged Japanese quails were loaded by attaching a tube weight corresponding to 12% of the bird's body weight for 7 or 21 days. Muscle from the contralateral side served as the intraanimal control. Relative to the intraanimal contralateral control side, the muscle wet weight increased by 96% in young birds, whereas the muscle weight gain in aged birds was not significant after 7 days of loading. After 21 days of loading, muscle weight significantly increased by 179% and 102% in young and aged birds, respectively. Heat shock protein (HSP)72 and HSP27 protein contents in the loaded sides were higher than on the control sides exclusively in young birds after 7 days of loading. Compared with the contralateral control muscle, the extent of apoptotic DNA fragmentation and the total cytosolic apoptosis-inducing factor protein content were reduced in all loaded muscles except for the 7-day-loaded muscles from the aged birds. Bax protein content was diminished in the loaded muscle relative to the control side from all groups, whereas Bcl-2 protein content was reduced in the young and aged muscles after 21 days of loading. The total cytosolic cytochrome c protein content was decreased and the X chromosome-linked inhibitor of apoptosis protein content was elevated in 7- and 21-day-loaded muscles relative to the intraanimal control muscle from young birds. Furthermore, after 7 days of loading the muscles of aged birds, H₂O₂ content and the total cytosolic protein content of second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point were elevated compared with the intraanimal control side. These data suggest that stretch overload-induced muscle hypertrophy is associated with changes in apoptosis in slow-tonic skeletal muscle. Moreover, discrepant apoptotic responses to muscle overload in young and aged muscles may account in part for the age-related decline in the capability for muscle hypertrophy. aging; sarcopenia; Bcl-2; Bax; heat shock proteins; apoptosis-inducing factor     

Collagen metabolism in mouse lung after X irradiation

Collagen and total protein synthesis rates have been determined in the lungs of CBA mice irradiated with single doses of X rays between 8 and 16 Gy. Mice were injected with [3H]proline accompanied by a large dose of unlabeled proline, and synthesis rates were measured at 2-month intervals from 8 to 31 weeks after irradiation. At 2 months after radiation treatment, collagen and total protein synthesis rates were significantly depressed but they had recovered by 4 months. By 6 months collagen synthesis rates had increased above control in a dose-dependent manner, so that in the 14-Gy dose group the fractional synthesis rate for collagen was 4.6 times higher than in control mice as measured by incorporation of [3H]proline. However, a significant net accumulation of collagen was seen only in the lungs of the highest dose group at 31 weeks, as indicated by total hydroxyproline measurements. There was a slight increase in the ratio of types I and III collagen. Late radiation damage in the CBA mouse lung is characterized by increased collagen metabolism, which may or may not lead to a net accumulation of collagen.     

Increased Collagen Synthesis Rate during Wound Healing in Muscle

Wound healing in muscle involves the deposition of collagen, but it is not known whether this is achieved by changes in the synthesis or the degradation of collagen. We have used a reliable flooding dose method to measure collagen synthesis rate in vivo in rat abdominal muscle following a surgical incision. Collagen synthesis rate was increased by 480% and 860% on days 2 and 7 respectively after surgery in the wounded muscle compared with an undamaged area of the same muscle. Collagen content was increased by approximately 100% at both day 2 and day 7. These results demonstrate that collagen deposition during wound healing in muscle is achieved entirely by an increase in the rate of collagen synthesis.