Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

Background

Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.

Methodology/Principal Findings

ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r² = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.

Conclusions/Significance

We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.     

The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass.

The effects of growth-suppressing and muscle-wasting treatments on muscle protein turnover and amino acid concentrations were determined in vivo. All treatments depressed protein synthesis and some treatments depressed protein breakdown. Only prolonged starvation increased protein breakdown. Muscle protein mass is regulated primarily through alterations in protein synthesis in all except emergency conditions. The increased concentrations of the branched-chain amino acids indicate that they are unlikely to be involved in this regulation.     

Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.     

Effect of combined aerobic and strength exercises on the regulation of mitochondrial biogenesis and protein synthesis and degradation in human skeletal muscle

We tested the hypothesis that strength exercise after intermittent aerobic exercise might activate signaling pathways that regulate mitochondrial biogenesis (activation of the AMPK and p38 pathways; the expression of PGC-1α, NT-PGC-1α, TFAM, and VEGFA mRNA), protein synthesis (phosphorylation level of p70S6K1^Thr389 and eEF2^Thr56; the expression IGF-1Ea, IGF-1Ec (MGF), and REDD1 mRNA) and proteolysis (phosphorylation level of FOXO1^Ser256; the expression of MURF1, MAFbx, and Myostatin mRNA) in trained skeletal muscles. Nine amateur endurance-trained athletes performed an intermittent aerobic cycling (70 min), followed by one-leg strength exercise (ES: four sets of knee extensions till exhaustion), while the other leg was resting (E). Gene expression and protein level were evaluated in samples from m. vastus lateralis taken before the exercise, 40 min, 5 and 22 h after the aerobic exercise. The phosphorylation level of the АСС^Ser79/222 (an endogenous marker of AMPK activity) and the expression of PGC-1α-related gene TFAM (a marker of mitochondrial biogenesis) were increased after E exercise and did not changed after ES exercise. The expression of PGC-1α and truncated isoform NT-PGC-1α was increased in both legs as well. Insulin concentration in blood was decreased significantly (7.5-fold) after aerobic exercise; the phosphorylation level of FOXO^Ser256 (a regulator of ubiquitin-related proteolysis) was decreased in both legs, which means that it was activated in both types of exercises; at the same time, the expression of the E3-ubiquitin ligase gene MURF1, its target, was only increased after E exercise. Neither aerobic or combined exercise had a significant effect on the regulation of protein synthesis: there were no changes in either expression of IGF-1Ea and IGF-1Ec(MGF) mRNA isoforms or the phosphorylation levels of markers of protein synthesis p70S6K1^Thr389 and eEF2^Thr56. Thus, the performance of strength exercise immediately after aerobic one prevented the activation of mitochondrial biogenesis in endurance-trained muscles: activation of AMPK pathway and the expression of TFAM are decreased, while protein synthesis regulation is not affected. At the same time, the strength exercise inhibited the expression of MURF1 gene (a marker of ubiquitin proteasome system), which was induced by aerobic exercise. We suggest that strength exercise performed immediately after intense intermittent aerobic exercise may have a negative effect on aerobic performance if used chronically.     

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.     

Effects of corticosterone on connectin content and protein breakdown in rat skeletal muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

L-Carnitine: a potential treatment for blocking apoptosis and preventing skeletal muscle myopathy in heart failure

Skeletal muscle in congestive heartfailure is responsible for increased fatigability and decreasedexercise capacity. A specific myopathy with increased expression offast-type myosins, myocyte atrophy, secondary to myocyteapoptosis triggered by high levels of circulating tumornecrosis factor- (TNF-) has been described. In an animal model ofheart failure, the monocrotaline-treated rat, we have observed anincrease of apoptotic skeletal muscle nuclei. Proapoptoticagents, caspase-3 and -9, were increased, as well as serum levels ofTNF- and its second messenger sphingosine. Treatment of rats withL-carnitine, known for its protective effect on musclemetabolism injuries, was found to inhibit caspases and to decrease thelevels of TNF- and sphingosine, as well as the number ofapoptotic myonuclei. Staurosporine was used in in vitro experimentsto induce apoptosis in skeletal muscle cells in culture. WhenL-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that L-carnitine can preventapoptosis of skeletal muscles cells and has a role in thetreatment of congestive heart failure-associated myopathy.

     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D-myo-[2-3H]inositol, addition of alpha-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of alpha-antagonists (phentolamine, prazosin). beta-Agonists and/or beta-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of beta-receptor mediated processes was suggested by the beta-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under alpha-adrenergic control.     

Combined effects of hyperaminoacidemia and oxandrolone on skeletal muscle protein synthesis

We investigated whether the normal anabolic effects of acute hyperaminoacidemia were maintained after 5 days of oxandrolone (Oxandrin, Ox)-induced anabolism. Five healthy men [22 +/- 3 (SD) yr] were studied before and after 5 days of oral Ox (15 mg/day). In each study, a 5-h basal period was followed by a 3-h primed-continuous infusion of a commercial amino acid mixture (10% Travasol). Stable isotopic data from blood and muscle sampling were analyzed using a three-compartment model to calculate muscle protein synthesis and breakdown. Model-derived muscle protein synthesis increased after amino acid infusion in both the control [basal control (BC) vs. control + amino acids (C+AA); P < 0.001] and Ox study [basal Ox (BOx) vs. Ox + amino acids (Ox+AA); P < 0.01], whereas protein breakdown was unchanged. Fractional synthetic rates of muscle protein increased 94% (BC vs. C+AA; P = 0.01) and 53% (BOx vs. Ox+AA; P < 0.01), respectively. We conclude that the normal anabolic effects of acute hyperaminoacidemia are maintained in skeletal muscle undergoing oxandrolone-induced anabolism.