Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Iron supplementation is sufficient to rescue skeletal muscle mass and function in cancer cachexia

Cachexia is a wasting syndrome characterized by devastating skeletal muscle atrophy that dramatically increases mortality in various diseases, most notably in cancer patients with a penetrance of up to 80%. Knowledge regarding the mechanism of cancer‐induced cachexia remains very scarce, making cachexia an unmet medical need. In this study, we discovered strong alterations of iron metabolism in the skeletal muscle of both cancer patients and tumor‐bearing mice, characterized by decreased iron availability in mitochondria. We found that modulation of iron levels directly influences myotube size in vitro and muscle mass in otherwise healthy mice. Furthermore, iron supplementation was sufficient to preserve both muscle function and mass, prolong survival in tumor‐bearing mice, and even rescues strength in human subjects within an unexpectedly short time frame. Importantly, iron supplementation refuels mitochondrial oxidative metabolism and energy production. Overall, our findings provide new mechanistic insights in cancer‐induced skeletal muscle wasting, and support targeting iron metabolism as a potential therapeutic option for muscle wasting diseases.     

Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.     

Murine models of atrophy,cachexia, and sarcopenia in skeletal muscle

With the extension of life span over the past several decades, the age-related loss of muscle mass and strength that characterizes sarcopenia is becoming more evident and thus, has a more significant impact on society. To determine ways to intervene and delay, or even arrest the physical frailty and dependence that accompany sarcopenia, it is necessary to identify those biochemical pathways that define this process. Animal models that mimic one or more of the physiological pathways involved with this phenomenon are very beneficial in providing an understanding of the cellular processes at work in sarcopenia. The ability to influence pathways through genetic manipulation gives insight into cellular responses and their impact on the physical expression of sarcopenia. This review evaluates several murine models that have the potential to elucidate biochemical processes integral to sarcopenia. Identifying animal models that reflect sarcopenia or its component pathways will enable researchers to better understand those pathways that contribute to age-related skeletal muscle mass loss, and in turn, develop interventions that will prevent, retard, arrest, or reverse this phenomenon. This article is part of a Special Issue entitled: Animal Models of Disease.     

Myosin degradation fragments in skeletal muscle

Myosin heavy chain degradation fragments produced in vivo have been identified in chicken pectoralis muscle. The fragments were identified by electrophoresis of unfractionated extracts of chicken pectoralis muscle on sodium dodecyl sulfate/polyacrylamide gels followed by immunoblotting on nitrocellulose sheets. Monoclonal antibodies directed against the S2 and light meromyosin subfragments as well as type II myosin-specific polyclonal antibodies directed against the entire myosin heavy chain were used to characterize the fragments, which range in molecular weight from approximately 80,000 to 180,000. All fragments contain the extreme carboxy-terminal portion of the molecule and are distinct from the classical proteolytic fragments such as heavy and light meromyosin, S1, S2 or rod. These fragments appear to be produced in vivo by proteolytic cleavage of peptides from the amino-terminal (S1) end of the heavy chain while the myosin molecule is still embedded in the thick filament. Fragment concentrations are estimated to be approximately 5 to 10% of that of the intact myosin heavy chain. These fragments are not the result of artifactual damage to myosin, e.g. proteolysis or hydrodynamic shear. The techniques described in this paper provide a probe into the early stages of myosin and thick filament degradation in vivo.     

Mechanisms of experimental cancer cachexia. Interaction between mononuclear phagocytes and colon-26 carcinoma and its relevance to IL-6-mediated cancer cachexia.

In a recent report we showed that IL-6 is an important mediator of experimental cancer cachexia in the colon-26 (C-26) tumor system. In culture, on a per cell basis, C-26.IVX cell line (which develops tumors and induces severe cachexia of syngeneic hosts) produces up to 60-fold less IL-6 than single cell suspensions prepared from freshly excised tumors. In this study, the mechanism behind this observation was investigated. Analysis of the cellular composition of progressing C-26 tumors indicated they contained up to 6% of macrophages. T cells, B cells, and granulocytes were not detected in the tumors. Because C-26.IVX line grown in vitro contained no macrophages, the possibility that macrophage products may augment IL-6 synthesis by the tumor cells was tested. Indeed, IL-1 beta in a dose-dependent manner and at picogram amounts could potentiate IL-6 production by the C-26 cell line. The presence of high affinity receptors for IL-1 on the C-26.IVX cell line was established. These cells expressed approximately 1500 IL-1 sites per cell with a dissociation constant of approximately 20 pM. Next, we attempted to mimic the situation in vivo by coculture of C-26.IVX cells with syngeneic peritoneal macrophages and found that this condition gives rise to an augmented IL-6 production similar to that observed with in vivo derived tumor cells or rIL-1 beta-treated C-26.IVX cells. Furthermore, anti-IL-1 type I receptor antibody completely blocked C-26.IVX IL-6 production induced by either rIL-1 beta or by peritoneal macrophages. Taken together, these data suggest a pathway of IL-6 production by C-26 tumors that involves a cellular interaction between IL-1R-expressing tumor cells and host-derived macrophages. The results also suggest that this interaction significantly contributes to cachectic events endured by the tumor-bearing host.     

JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C(2)C(12) myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia.     

Insulin degradation by human skeletal muscle

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 × g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100 000×g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the ¹²⁵I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a K_m of 1.63·10^?7 M (±0.32) and was competitively inhibited by proinsulin and glucagon with K_i values of 2.1 · 10?⁶ M and 4.0 · 10^?6 M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120 000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [¹²⁵I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.     

Hormonal regulation of protein degradation in skeletal and cardiac muscle

A number of hormones produce either anabolic or catabolic effects on protein degradation in muscle. These effects can account for the changes in muscle proteolysis associated with a variety of physiological and pathological states. Thus the balance of hormones within the organism seems to play an important role in the overall regulation of this process. In the fed state, insulin may be the single most important factor maintaining low rates of proteolysis, whereas the catabolic effects of the glucocorticoid hormones in fasting seem to predominate. The proportions of these hormones may be important not only during starvation, but also in trauma and in diseases associated with their altered production and secretion (e.g., diabetes, Cushing's syndrome). Hyperthyroidism too causes catabolic effects on muscle proteolysis.     

Specificity of the effects of leucine and its metabolites on protein degradation in skeletal muscle.

The effects of leucine, its metabolites, and the 2-oxo acids of valine and isoleucine on protein synthesis and degradation in incubated limb muscles of immature and adult rats were tested. Leucine stimulated protein synthesis but did not reduce proteolysis when leucine transamination was inhibited. 4-Methyl-2-oxopentanoate at concentrations as low as 0.25 mM inhibited protein degradation but did not change protein synthesis. The 2-oxo acids of valine and isoleucine did not change protein synthesis or degradation even at concentrations as high as 5 mM. 3-Methylvalerate, the irreversibly decarboxylated product of 4-methyl-2-oxopentanoate, decreased protein degradation at concentrations greater than or equal to 1 mM. This was not due to inhibition of 4-methyl-2-oxopentanoate catabolism, because 0.5 mM-3-methylvalerate did not suppress proteolysis, even though it inhibited leucine decarboxylation by 30%; higher concentrations of 3-methylvalerate decreased proteolysis progressively without inhibiting leucine decarboxylation further. During incubation with [1-14C]- and [U-14C]-leucine, it was found that products of leucine catabolism formed subsequent to the decarboxylation of 4-methyl-2-oxopentanoate accumulated intracellularly. This pattern was not seen during incubation with radiolabelled valine. Thus, the effect of leucine on muscle proteolysis requires transamination to 4-methyl-2-oxopentanoate. The inhibition of muscle protein degradation by leucine is most sensitive to, but not specific for, its 2-oxo acid, 4-methyl-2-oxopentanoate.     

Leucine degradation in cell-free extracts of skeletal muscle.

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.     

Post-mortem timing of skeletal muscle biochemical and mechanical degradation

Fresh cadaveric human tissue is a valuable resource that is used to address important clinical questions. However, it is unknown how post-mortem time impacts skeletal muscle mechanical and biochemical properties. We simulated morgue conditions in rabbits and tested the passive mechanical properties of muscle bundles, and the degradation of myosin heavy chain, collagen, and titin at specific intervals up to 7 days post-mortem. While a great deal of inter-specimen variability was observed, it was independent of post-mortem time. Passive mechanics, myosin heavy chain, and collagen content were all unaffected while the titin protein degraded up to 80% over 7 days post-mortem. These data indicate that fresh cadaveric tissue may be used for passive mechanical testing and that certain biochemical properties are unchanged up to 7 days after death.     

The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+) mouse

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+) mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+) mouse is not known. Cachexia progression was studied in Apc(Min/+) mice that were either weight stable (WS) or had initial (≤5%), intermediate (6-19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ～50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.     

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.     

Ketone bodies inhibit leucine degradation in chick skeletal muscle

1. DL-beta-hydroxybutyrate (4 mM) increased the net rate of leucine transamination and the net rate of 2-oxoisocaproate (KIC) production in extensor digitorum communis muscles from fed chicks. 2. DL-beta-hydroxybutyrate at 1 and 4 mM inhibited leucine oxidative decarboxylation in muscles from fed chicks. 3. Acetoacetate at 1 and 4 mM inhibited leucine oxidative decarboxylation and total leucine oxidation, but increased net KIC production in muscles from fed chicks. 4. Both DL-beta-hydroxybutyrate and acetoacetate at 1 and 4 mM inhibited the net rate of leucine transamination and the rates of leucine oxidative decarboxylation and total leucine oxidation in muscles from 24-hr fasted chicks.     

Early development of protein metabolic perturbations in the liver and skeletal muscle of tumour-bearing rats. A model system for cancer cachexia.

In rats into which a fast-growing ascites hepatoma (Yoshida AH-130) had been transplanted, tumour growth elicited a marked loss of body weight until the animal's death in about 2 weeks. Overall tissue protein metabolism was simultaneously studied in vivo in the gastrocnemius muscle and liver after labelling with [14C]bicarbonate. Early and progressive atrophy developed in the gastrocnemius muscle, the underlying metabolic imbalance being expressed by an elevation in the apparent protein-degradation rate, with no changes in the apparent synthesis rate. A transient hyperplastic response preceded waste in the liver, both states being associated with alterations in protein-degradation rate: an initial decrease during liver growth, then an acceleration as liver regressed. Protein-synthesis rates, virtually unchanged during liver growth, were elevated in the subsequent phase, although not sufficient to balance the enhanced breakdown. Thus, in the tumour host tissues examined, altered states of protein turnover appeared to result mostly from changes in rates of protein breakdown. In sharp contrast with the negative protein balance in the host, the ascites hepatoma cells had the ability to grow or at least, in advanced stages, to maintain a stationary state.     

Ghrelin improves body weight loss and skeletal muscle catabolism associated with angiotensin II-induced cachexia in mice

Ghrelin is a gastric peptide that regulates energy homeostasis. Angiotensin II (Ang II) is known to induce body weight loss and skeletal muscle catabolism through the ubiquitin-proteasome pathway. In this study, we investigated the effects of ghrelin on body weight and muscle catabolism in mice treated with Ang II. The continuous subcutaneous administration of Ang II to mice for 6days resulted in cardiac hypertrophy and significant decreases in body weight gain, food intake, food efficiency, lean mass, and fat mass. In the gastrocnemius muscles of Ang II-treated mice, the levels of insulin-like growth factor 1 (IGF-1) were decreased, and the levels of mRNA expression of catabolic factors were increased. Although the repeated subcutaneous injections of ghrelin (1.0mg/kg, twice daily for 5days) did not affect cardiac hypertrophy, they resulted in significant body weight gains and improved food efficiencies and tended to increase both lean and fat mass in Ang II-treated mice. Ghrelin also ameliorated the decreased IGF-1 levels and the increased mRNA expression levels of catabolic factors in the skeletal muscle. IGF-1 mRNA levels in the skeletal muscle significantly decreased 24h after Ang II infusion, and this was reversed by two subcutaneous injections of ghrelin. In C2C12-derived myocytes, the dexamethasone-induced mRNA expression of atrogin-1 was decreased by IGF-1 but not by ghrelin. In conclusion, we demonstrated that ghrelin improved body weight loss and skeletal muscle catabolism in mice treated with Ang II, possibly through the early restoration of IGF-1 mRNA in the skeletal muscle and the amelioration of nutritional status.