Evidence for degradation of myofibrillar proteins in lysosomes. Myofibrillar proteins derivatized by intramuscular injection of N-ethylmaleimide are sequestered in lysosomes.
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Actomyosin↗
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Expression of the stress proteins, ubiquitin, heat shock protein 72, and myofibrillar protein content after 12 weeks of leg cycling in persons with spinal cord injury.
OBJECTIVE: To determine the effects of leg cycling exercise on ubiquitin (UBI), heat shock protein 72 (HSP-72) mRNA, protein expression, and myofibrillar protein content in individuals with spinal cord injury (SCI). DESIGN: Case series. SETTING: Motor behavior laboratory. PARTICIPANTS: Seven subjects with motor-complete SCI (4 men, 3 women). INTERVENTION: A 12-week exercise program involving an electromagnetically braked recumbent bicycle ergometer, which allowed for passive exercise of the legs. Training occurred 2 days a week at approximately 75% of each subject's maximum heart rate. MAIN OUTCOME MEASURES: Total body mass (TBM) and muscle biopsies were obtained pre- and posttraining. The mRNA and protein expression of UBI, HSP-72, and myofibrillar protein content were determined. RESULTS: Nonsignificant increases (P > .05) of 2.45% were observed for TBM. There were significant increases (P < .05) in the expression of both HSP-72 mRNA (33.71%) and protein (30.23%). For UBI, there were also significant decreases (P < .05) in the expression of both mRNA (26.86%) and protein (69.43%). Myofibrillar protein content increased significantly (P < .05, 41.86%). CONCLUSION: Leg cycling exercise in SCI increases myofibrillar protein content, possibly because of up-regulation in the expression of HSP-72 with concomitant down-regulation in the expression of UBI.
Adolescent↗
Biochemical changes in myofibrillar protein isolates exposed to three oxidizing systems.
The objective of the study was to compare three different oxidizing systems commonly present in muscle foods for their influence on the biochemical properties of muscle proteins. Myofibrillar protein isolate (MPI) prepared from pork serratus ventralis muscle was suspended (30 mg protein/mL) in 15 mM piperazine-N,N-bis(2-ethane sulfonic acid) buffer (pH 6.0). Oxidation was induced by incubating the protein suspension at 4 degrees C for 24 h with (i) a hydroxyl radical-generating system (HRGS: 10 microM FeCl3, 0.1 mM ascorbic acid, and 0.05-5.0 mM H2O2), (ii) a lipid-oxidizing system (LOS: 0.05-5.0 mM linoleic acid and 3750 units of lipoxidase/mL), or (iii) a metmyoglobin-oxidizing system (MOS: 0.05-0.5 mM metmyoglobin). Changes in oxidized MPI were measured as Ca- and K-ATPase activities, formation of protein carbonyls and 2-thiobarbituric acid-reactive substances (TBARS), loss of protein thermal stability, and protein aggregation. The three oxidizing matrixes induced complex MPI changes; for example, the Ca- and K-ATPase activities were altered mainly by low-concentration oxidants, but the changes were unique for each oxidizing system. The carbonyl content in MOS-treated MPI was the highest, while the TBARS production, changes in thermal properties, and loss of the myosin heavy chain were the greatest in HRGS-treated MPIs. Overall, the hydroxyl radical-producing medium appeared to be the most oxidative to myofibrillar proteins under the experimental conditions employed in the study.
Adenosine Triphosphatases↗
F-Protein, a myofibrillar protein interacting with myosin.
F-Protein has an amino acid composition distinctively different from those of myofibriller proteins so far reported to be of similar chain weight: M-protein component II, alpha-actinin, and AMP deaminase. Its molecular weight was estimated to be 121,000 by sedimentation equilibrium in 0.3 M KCl, 10 mM potassium phosphate, pH 6.5. Its binding to myosin was inhibited by C-protein. It reduced the effect of C-protein on the assembly reaction of myosin in vitro.
Amino Acids↗
Diarrhea reduces the rates of cardiac protein synthesis in myofibrillar protein fractions in rats in vivo.
Although chronic diarrhea affects heart function and morphology, the pathogenic mechanisms are unknown. It was our hypothesis that diarrhea imposes metabolic stress to inhibit the synthesis of new contractile proteins. To test this hypothesis, we investigated the effects of lactose-induced diarrhea in rats. The groups were: 1) freely fed controls, 2) rats with lactose-induced diarrhea or 3) pair-fed rats. After 1 wk, hearts from the rats were subjected to subcellular fractionation techniques to isolate the major protein fractions, including myofibrillar proteins. The rates of protein synthesis were measured with concomitant assay of cardiac composition and plasma analytes. In comparison with the control group, diarrhea induced the following changes (P < 0.05): a decrease in heart weight, reduced RNA and mixed protein contents and a reduction in the fractional rate of mixed protein synthesis. There was a reduction in the content of all protein fractions. The fractional synthesis rate was reduced only for the myofibrillar fraction. Plasma insulin-like growth factor-I, but not corticosterone, was reduced. Plasma cholesterol and triglyceride concentrations were also reduced. In comparison with the pair-fed group, diarrhea induced the following changes (P < 0.05): a reduction in heart weight and fractional rate of mixed protein synthesis, reduced myofibrillar absolute synthesis rate and increased sarcoplasmic/myofibrillar fractional synthesis rate ratio. Plasma bicarbonate, triglyceride and urea concentrations were reduced, with an increase in albumin. Diarrhea impaired cardiac biochemistry, including a reduction in protein content and synthesis. A substantial proportion of these changes is due to anorexia, but the selective reduction in the synthesis of contractile proteins is a feature exclusive to the diarrhea group and may be due to reductions in plasma insulin-like growth factor-I.
Animals↗
Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and the myofibrillar structure.
1. The mode of degradation of myofibrillar proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not alpha-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,000, 150,000, 87,000, 81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,000 and 30,000 Da. Troponin T was degraded into fragments of 21,000, 12,000 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myofibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.
Animals↗
Effect of protein and protein-free energy intake on myofibrillar protein degradation in preruminant calves of 120 and 200 kilograms of live weight.
We studied the effect of protein and protein-free energy intake on the degradation rate of myofibrillar protein in preruminant calves in two experiments. Both experiments were similar in design but were performed with two live weight ranges: 80 to 160 kg (Exp. 1) and 160 to 240 kg (Exp. 2). In each experiment, calves were allocated to either an initial slaughter group or to one of 12 treatments (three calves per treatment), which consisted of six protein intake levels at each of two protein-free energy intake levels. Calves were slaughtered and analyzed for body composition when they had reached the target weight. A balance trial was performed when calves had reached 120 and 200 kg BW in Exp. 1 and 2, respectively. Myofibrillar protein degradation rate was measured by the urinary excretion of 3-methylhistidine. Correction of 3-methylhistidine excretion for myofibrillar protein mass was performed based either on the urinary creatinine excretion rate or on estimated body composition during the balance trial. In both experiments, fractional rates of myofibrillar protein degradation increased with increasing protein intake (P < .01). Fractional degradation rates increased with increasing protein-free energy intake in Exp. 1 (P < .10) and in Exp. 2 (P < .05). We conclude that the increased protein deposition rates, reported earlier, induced by nutrient intake are accompanied by increased myofibrillar protein degradation rates. Furthermore, we consider urinary creatinine excretion to be unreliable as an estimator of myofibrillar protein mass in experiments in which a large range of nutrient intakes is applied.
Animals↗
Effects of protein deficiency on muscle myofibrillar protein turnover in adult rats.
The rates of gain, catabolism, synthesis and reutilization of myofibrillar protein were measured in adult rats fed a protein-free diet, low protein diet (2% lactalbumin) or control diet (10% lactalbumin) for 14 to 31 days. Two forms of synthesis were measured: exogenous synthesis (nitrogen derived from diet) and endogenous synthesis (nitrogen derived from catabolized body protein). The rate of gain of myofibrillar protein was measured as the rate of increase in its weight and the rate of catabolism was determined from urinary 3-methylhistidine excretion. The rate of total synthesis was calculated as the sum of these two rates. Exogenous synthesis was calculated from the recovery of isotope in protein 24 h after oral administration of 15N-leucine and endogenous synthesis was calculated as the difference between the total synthesis and exogenous synthesis. Reutilization was calculated as the ratio of endogeneous synthesis to catabolism. The rate of catabolism was slightly decreased in protein deficiency (2.1, 2.1 and 2.6% in the protein-free, low protein and control groups, respectively), while that of synthesis was significantly decreased in protein deficiency (1.3, 2.0 and 3.3% in the respective groups). Restriction of protein intake resulted in a decrease in the rate of exogenous synthesis, without appreciable change of endogenous synthesis. The reutilization rate of endogenous N was estimated to be about 70% in rats with restricted protein intakes and about 50% in those with a normal protein intake.
Animals↗
Myofibrillar protein turnover. Synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation.
Measurement of rates of synthesis of skeletal-muscle proteins in adult rats shows that the faster overall rate of turnover in diaphragm and soleus muscles compared with several other, more glycolytic, muscles is also exhibited by the myofibrillar proteins, since the ratio of sarcoplasmic to myofibrillar protein synthesis is similar for all muscles. Further, throughout postnatal development, when the overall turnover rate falls with age, parallel changes occur for the myofibrillar proteins, as indicated by a constant ratio of sarcoplasmic to myofibrillar protein synthesis (2.06) in the steady state after overnight starvation. Only in the youngest (4 weeks old) rats is a slightly lower ratio observed (1.72). These results indicate that, when changes in the overall turnover rate of muscle proteins occur, the relative turnover of the two major protein fractions stays constant. However, measurements in the non-steady state during growth and after starvation for 4 days show that the relative synthesis rates of the two fractions change as a result of a disproportionate increase in myofibrillar protein synthesis during growth and decrease during starvation. Thus the synthesis rate of the slower-turning-over myofibrillar protein fraction is more sensitive to nutritional state than is that of the sarcoplasmic protein. It is suggested that such responses may help to maintain constant tissue composition during non-steady-state conditions of growth and atrophy.
Animals↗
Suppression of myofibrillar protein degradation after refeeding in young and adult mice.
A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation after refeeding in young rats and mice. However, it is unclear whether this suppression is seen in adult animals. This study was undertaken to compare dietary protein-induced suppression of myofibrillar protein degradation in young and adult mice. Reductions in rates of myofibrillar protein degradation measured by N-methylhistidine (MeHis) released from the isolated extensor digitorum longus muscle were found at 4 to 7 h after refeeding in both young (7-wk-old) and adult (8-mo-old) mice, indicating that the response time of feeding-induced suppression of myofibrillar protein degradation was the same. When young (8-wk-old) mice were fed a 20% casein diet (20C) for 1 h after 18 h starvation, the rate of myofibrillar protein degradation was significantly decreased at 4 h after refeeding; however, mice fed a 10% casein diet (10C), 5% casein diet (5C), or protein-free diet (0C) did not show suppression of myofibrillar protein degradation. Adult (8-mo-old) mice fed 20C or 10C showed a reduction in the rate of MeHis release. The plasma concentration of leucine in young mice was only higher when they were fed 20C. Adult mice fed 20C or 10C showed higher plasma concentrations of leucine. These results suggest that postprandial suppression of myofibrillar protein degradation occurs in adult mice as in young mice, but the adult mice respond to a lower amount of dietary casein compared to the young mice.
Animals↗
Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium.
We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.
Animals↗
Effect of the thyroid status and protein-calorie malnutrition on the rate of myofibrillar protein degradation in mature male rats.
The urinary excretion of N tau-methylhistidine (3-methylhistidine: 3-Mehis), an index of the rate of myofibrillar protein catabolism, was determined in intact and thyroid ectomized mature male rats, receiving intraperitoneally either vehicle (0.9% NaCl) or thyroxine (T4) replacement (2 microgram/100 g body weight/day) during 20 days. Rats were fed either an adequate control of a low-protein low-energy diet. In addition, body weight changes and food intake were recorded throughout the experiment. At the end of the 20-day period, livers and several muscles from hind limbs were excised and weighed. A sample of blood was then taken for serum insulin, triiodothyronine (T3) and thyroid-stimulating hormone (TSH) determination. As compared to the well-nourished animals, a significant (p less than 0.05) reduction in the rate of growth, food intake, 3-Mehis and serum insulin and T3 concentrations was observed in the rats fed the low-protein low-energy diet. In both dietary groups, thyroidectomy increased serum TSH levels and tended to reduce 3-Mehis output and liver and muscle sizes, although there was a different response according to the type of muscle excised. T4 replacement improved growth and restored T3 levels, especially in the well-fed animals, but it failed to restore either serum insulin concentrations or 3-Mehis output in either dietary groups. In conclusion, both thyroidectomy and protein-calorie malnutrition reduced the rate of myofibrillar protein breakdown in the mature rat and T4 replacement had no effect in restoring the normal range of myofibrillar protein degradation.
Animals↗
Protein synthesis, myosin ATPase activity and myofibrillar protein composition in hearts from tumour-bearing rats and mice.
Growing rats and adult weight-stable mice bearing a transplantable methylcholanthrene-induced sarcoma were compared with animals with various states of malnutrition. Heart protein synthesis was measured in vivo. Myocardial RNA, myofibrillar protein composition and the Ca2+-activated ATPase activity in heavy chains of native myosin were measured. 'Fingerprints' were made from myosin by trypsin treatment to evaluate possible structural changes in the protein. Cardiac protein-synthesis rate was decreased by 20% in growing tumour-bearing rats, by 35% in protein-malnourished (rats) and by 47% in starved rats, compared with freely fed controls (P less than 0.05). Adult tumour-bearing mice showed no significant decrease in myocardial protein synthesis. Pair-weighed control mice had significantly depressed heart protein synthesis. Protein translational efficiency was maintained in both tumour-bearing rats and mice, but was decreased in several groups of malnourished control animals. The Ca2+-activated myosin ATPase activity was decreased in all groups of malnourished animals, including tumour-bearing mice and rats, without any evidence of a change in cardiac isomyosin composition. We conclude that loss of cardiac muscle mass in tumour disease is communicated by both depressed synthesis and increased degradation largely owing to anorexia and host malnutrition. Increased adrenergic sensitivity in hearts from tumour-bearing and malnourished animals is not communicated by increased Ca2+-activated ATPase activity. This may be down-regulated in all groups with malnutrition, without any observable alterations in the isomyosin profile.
Animals↗
Myofibrillar protein turnover in cardiac hypertrophy due to aortic regurgitation.
We recently demonstrated that total cardiac protein and myosin heavy chain fractional synthesis rates were not increased during the progressive cardiac hypertrophy that occurred 1 month following induction of aortic regurgitation. The increase in total cardiac protein and myosin heavy chain observed after 1 month of chronic volume overload was caused by a decrease in protein fractional degradation rates. The objective of the present study was to determine in vivo the relative contributions of protein synthesis and degradation of a variety of individual myofibrillar protein constituents, other than myosin heavy chain, to the left ventricular hypertrophic response to chronic aortic regurgitation. Intravenous infusions of [3H]-leucine were administered 3 days and 1 month following surgical induction of aortic regurgitation and sham operation in rabbits, and actin, myosin light chains 1 and 2, alpha-actinin and desmin fractional synthesis rates were obtained by analysis of plasma and protein hydrolysate data using [14C]-dansyl chloride assays. Individual myofibrillar protein growth rates were determined from protein concentration and serial echocardiographic and postmortem left ventricular weight measurements; protein degradation rates were determined by subtraction of growth rates from synthesis rates. Individual myofibrillar protein content increased most rapidly during the 1st week and progressively increased at a slower rate between 1 week and 1 month, in parallel with increases in left ventricular weight. In comparison with sham-operated controls, individual myofibrillar protein fractional synthesis rates were consistently increased at 3 days but not at 1 month. Progressive myocyte hypertrophy occurring at 1 month was caused by a decrease in myofibrillar protein fractional degradation rates. Increased myofibrillar protein synthesis contributed only to the early phase of myocyte hypertrophy while progressive hypertrophy in chronic aortic regurgitation was due to suppression of myofibrillar protein degradation.
Actins↗
The possible relevance of autoxidative glycosylation in glucose mediated alterations of proteins: an in vitro study on myofibrillar proteins.
The present work was carried out to examine the role of glycation and transition metal catalysed autoxidation of sugars in glucose-mediated alterations of myofibrillar proteins. Myofibrils were prepared from rat skeletal muscle and incubated with 1) sugar alone 2) sugar and micromolar concentrations of transition metals (Cu2+ or Fe3+) 3) transition metals alone and the control remained without sugar or transition metals. A significant increase in extent of glycation and decrease in ATPase activity of myofibrils incubated under autoxidative conditions were observed over the other three incubations. Reducing agent 2-mercaptoethanol was highly effective in preventing the alterations induced by glucoxidation, compared to EDTA and aminoguanidine, suggesting the involvement of thiol group oxidation in the reduced function of the protein. Free radical scavengers like catalase, benzoic acid and mannitol were also effective in preventing glucose mediated alterations. Although a high concentration of glucose alone has an insignificant effect on myofibrils in vitro, the results from the present work suggest that glucose in combination with transition metals could lead to functional alterations of myofibrils, and this process by generating free radicals may contribute to the overall complications of diabetes and aging.
Animals↗
Measurements of myofibrillar protein breakdown in newborn human infants.
1. Myofibrillar protein breakdown was calculated from the urinary excretion ratio of NT- methylhistidine (3-methylhistidine) to creatinine in newborn premature and full-term infants. Representative values were obtained from single voidings provided that the infant's metabolic status was stable. 2. NT- Methylhistidine in infant urine was measured by a rapid Auto Analyser method and shown to give similar values to those obtained by ion-exchange separation techniques. 3. The molar excretion ratio of NR- methylhistidine to creatinine averaged 0.0159 in urine samples obtained within 12 h after birth. A similar ratio was found in amniotic fluid collected at birth. It is argued that this ratio does not reflect a low rate of myofibrillar protein breakdown in the foetus, but rather a more effective transplacental passage of NT- methylhistidine than of creatinine. 4. The urinary ratio increased during the first 2 days after birth to a plateau at 0.0372. This represents a myofibrillar protein degradation rate of 3.40% day-1 in full-term infants. 5. The molar excretion ratio during the period 40-120 h after birth increased in premature infants and reflects a fractional degradation rate of 5.34% day-1 in those infants weighting less than 1 kg at birth. 6. Lower excretion ratios were found in some infants of diabetic mothers and in athyroid infants. 7. The urinary excretion ratio of NT-methylhistidine to creatinine is presented as a useful method for evaluating the breakdown rate of myofibrillar protein in neonates and can be applied to a number of abnormal nutritional or hormonal states.
Amniotic Fluid↗
Molecular diversity of myofibrillar proteins: gene regulation and functional significance.
Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.
Animals↗
Myofibrillar protein synthesis in young and old men.
We tested the hypothesis that healthy older men (> 60 yr old) have a slower rate of myofibrillar protein synthesis than young men (< 35 yr old). Myofibrillar protein synthesis was determined by the in vivo incorporation of L-[1-13C]leucine into myofibrillar proteins obtained by muscle biopsy. Subjects were eight young (21-31 yr) and eight older (62-81 yr) men, all healthy and moderately active. There was no significant difference in the mean height and weight of the two age groups, but the older group had 12% less lean body mass (determined by 40K counting) and 21% less muscle mass (estimated by urinary creatinine excretion). Upper leg strength was approximately one-third lower in the older subjects according to isokinetic dynamometry. The fractional rate of myofibrillar protein synthesis was 28% slower in the older group (0.039 +/- 0.009 vs. 0.054 +/- 0.010 %/h, mean +/- SD, P < 0.01). Total myofibrillar protein synthesis, estimated as total myofibrillar mass (from creatinine excretion) times the fractional synthesis rate, was 44% slower in the older group (1.4 vs. 2.5 g/h, P < 0.001). Whole body protein synthesis, assessed as the difference between leucine disappearance rate and leucine oxidation, was marginally slower (8%, P = 0.10) in the older group, but not when the data were adjusted for lean body mass. Myofibrillar protein synthesis was a smaller fraction of whole body protein synthesis in the older group (12 vs. 19%). Reduced myofibrillar protein synthesis may be an important mechanism of the muscle atrophy associated with aging.
Adult↗