The proteomics of ageing

Proteomics provides an extremely powerful tool for the study of variations in protein expression between individuals, different disease states and different conditions. One of the major challenges facing the medical profession in the forthcoming decades is to understand the changes that occur in individuals as they become older and to attempt to develop means to improve the quality of life for the otherwise healthy ageing population. The present study describes the first phase of such an investigation in which the andaged protein composition of human skeletal muscle sample from young and aged subjects are compared. These results provide the beginning of a Human Aged Skeletal Muscle Profile reference map which is an essential first step to further investigations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Change in fibre size, not number, in ageing skeletal muscle

Skeletal muscle fibre loss and other changes were evaluated in 25 female rats aged 6-24 months. Soleus (SOL) and extensor digitorum longus (EDL) muscles were removed bilaterally. Left SOL and EDL muscles were digested in nitric acid, then, using a dissecting microscope, total fibre numbers for each muscle were obtained. Right muscles were frozen, sectioned and stained with ATPase and alkaline phosphatase to assess fibre-type distribution and capillary number. No significant change in total fibre number was seen with age in either muscle. Atrophy was common after age 12 months but was not found consistently. No reduction in capillary number was observed, even among atrophic fibres. Results suggest that atrophy and not fibre loss, per se, occurs with ageing and that atrophy is not due to capillary loss.     

Live strong and prosper: the importance of skeletal muscle strength for healthy ageing

Due to improved health care, diet and infrastructure in developed countries, since 1840 life expectancy has increased by approximately 2 years per decade. Accordingly, by 2050, a quarter of Europe’s population will be over 65 years, representing a 10 % rise in half a century. With this rapid rise comes an increased prevalence of diseases of ageing and associated healthcare expenditure. To address the health consequences of global ageing, research in model systems (worms, flies and mice) has indicated that reducing the rate of organ growth, via reductions in protein synthetic rates, has multi-organ health benefits that collectively lead to improvements in lifespan. In contrast, human pre-clinical, clinical and large cohort prospective studies demonstrate that ageing leads to anabolic (i.e. growth) impairments in skeletal muscle, which in turn leads to reductions in muscle mass and strength, factors directly associated with mortality rates in the elderly. As such, increasing muscle protein synthesis via exercise or protein-based nutrition maintains a strong, healthy muscle mass, which in turn leads to improved health, independence and functionality. The aim of this review is to critique current literature relating to the maintenance of muscle mass across lifespan and discuss whether maintaining or reducing protein synthesis is the most logical approach to support musculoskeletal function and by extension healthy human ageing.     

Molecular mechanisms and therapeutics of the deficit in specific force in ageing skeletal muscle

The age-related impairment in muscle force is only partially explained by the loss of muscle mass. The loss both in specific and absolute forces contributes to the muscle weakness measured in the elderly and in animal models of ageing. Successful interventions aimed at preventing age-associated functional deficits will require a better insight into the mechanisms underlying the decline in muscle-specific force. The present review article is focused on recent evidence supporting excitation-contraction uncoupling as a key factor underlying fast and slow muscle fiber impairment with ageing. The molecular, functional and structural factors supporting this theory and counteracting measures such as insulin-like growth factor 1 transgenic overexpression are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth factors and muscle ageing

Loss of muscle mass (sarcopenia) is one of the main problems associated with ageing as it has major health care as well as socioeconomic implications. The growth hormone (GH)/IGF-I axis is regarded as an important regulator of muscle mass. However, it is now appreciated that other tissues in addition to the liver express IGF-I and that there are local as well as systemic forms of IGF-I which have different functions. At least two different kinds of IGF-I that are expressed by skeletal muscle are derived from the IGF-I gene by alternative splicing, one of which is expressed in response to physical activity which has now been called 'mechano growth factor' (MGF). The other is similar to the systemic or liver type (IGF-IEa) and is important as the provider of mature IGF-I required for upregulating protein synthesis. MGF differs from systemic IGF-IEa in that it has a different peptide sequence which is responsible for replenishing the satellite (stem) cells in skeletal muscle. The ability to produce MGF declines with age, and this is commensurate with the decline in circulating GH levels. GH treatment up regulates the level of IGF-I gene expression in older people and when combined with resistance exercise more is spliced towards MGF and hence should improve the ability of muscle to respond to physical activity. The possibility of ameliorating sarcopenia using MGF is discussed.     

Rabbit skeletal muscle PO2 during hypodynamic sepsis

We measured skeletal muscle tissue PO2 (PtO2) in anesthetized rabbits (n = 7) following infusion of an intravenous bolus of E coli endotoxin. An array of surface PO2 microelectrodes was placed over the hindlimb biceps femoris muscle and sufficient readings were obtained to construct a PtO2 histogram. Changes in the histogram standard deviation were used to characterize micro-circulatory maldistribution. Systemic O2 consumption (VO2) was measured by the expired gas method. Cardiac output (Q) and systemic O2 transport (TO2) were calculated. Samples of arterial, right atrial (ra), and hindlimb venous blood, from a catheter placed in the infrarenal portion of the vena cava, were simultaneously obtained for measurement of blood gases and saturations. Following the administration of endotoxin, there were decreases in Q and TO2 of approximately 50 percent. The VO2 initially decreased 23 percent, but returned to baseline levels 30 minutes after endotoxin administration. Systemic O2 extraction ratio (ERO2 = VO2/TO2) increased from 0.32 +/- .03 to 0.54 +/- .07 (p less than 0.01), whereas hindlimb ERO2 increased from 0.42 +/- .03 to 0.60 +/- .02 (p less than 0.01). The arithmetic mean of the PtO2 histograms decreased after endotoxin infusion (43 +/- 4 to 7 +/- 2 mm Hg; p less than 0.01), but PLO2 remained at baseline levels (35 +/- 2 vs. 33 +/- 2 mm Hg; p = NS). The standard deviation of the PtO2 histograms remained constant during the experiment. This finding supports the notion that skeletal muscle microcirculatory heterogeneity does not increase during endotoxin induced hypodynamic sepsis.     

Urocortin II treatment reduces skeletal muscle mass and function loss during atrophy and increases nonatrophying skeletal muscle mass and function

Two corticotropin-releasing factor 2 receptor (CRF2R)-selective peptides have been recently described, urocortin II (also known as stresscopin-related peptide) and urocortin III (stresscopin). We have used urocortin II to evaluate the effects of activation of the CRF2R on skeletal muscle-related physiological processes. Administration of urocortin II to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, urocortin II treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. Finally, we observed increased skeletal muscle mass and force in normal muscles when mice are treated with urocortin II. These results were confirmed using two additional CRF2R agonists, urocortin I and sauvagine. Thus, activation of the CRF2R modulates skeletal muscle mass in both normal and atrophying muscle. Therefore, CRF2R-selective agonists may find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia.     

Structural dynamics of troponin during activation of skeletal muscle

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s⁻¹) matches the rate of Ca²⁺ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s⁻¹) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s⁻¹) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament–myosin head complex under physiological conditions.Contraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca²⁺ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2–4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5–7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca²⁺ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca²⁺ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).Open in a separate windowFig. 1.Troponin core complex with bifunctional probes on the muscle thin filament. On the left, the structure of the core complex of troponin from skeletal muscle in the Ca²⁺-saturated form (10) is shown, containing TnC (yellow) and parts of TnI (blue) and TnT (orange). BR probes cross-linked cysteines 56 and 63 (red spheres) along the C helix (green) in the TnC N lobe or cysteines 96 and 103 along the E helix (magenta) in the TnC C lobe. Black spheres indicate the Ca²⁺ regulatory sites and gray spheres the Ca²⁺/Mg²⁺ sites. On the right is the schematic representation of the troponin core complex oriented on the actin filament (light gray) according to the model of Knowles et al. (7). Each complex is anchored to one tropomyosin strand (dark gray) through the N terminus of TnT (orange arrow). Orange and blue sticks form the IT arm. The C terminus of TnI contains the two actin-binding regions (small blue rectangles), the switch peptide (blue triangle) and the remainder of the mobile domain (large blue rectangle). The position of the probes labeling C and E helices of TnC is marked by green and magenta rectangles respectively.A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg²⁺ as well as Ca²⁺. The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the “IT arm” (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lobe D helix in the Ca²⁺-bound crystal structure shown in Fig. 1, the D/E helix is broken in situ, as it is in the crystal structures of the Ca²⁺-bound cardiac isoform and the apo state of the skeletal isoform (9, 10). Thus, the C- and E-helix probes give independent information about the orientations of the TnC N lobe and the IT arm, respectively, in a muscle fiber.We separated the structural effects of Ca²⁺ and myosin binding during activation of demembranated muscle fibers in physiological conditions kinetically, using rapid jumps in intracellular [Ca²⁺] produced by photolysis of nitrophenyl-EGTA (NP-EGTA or caged Ca). Binding of Ca²⁺ to the regulatory sites of troponin is at least 10–20 times faster than myosin binding in the conditions used here, so we were able to resolve the kinetics of intermediate structural changes in the troponin signaling pathway, and relate them to those of calcium binding to troponin, myosin binding to actin, and force generation. We used three additional protocols to assess the role of myosin binding in muscle regulation in physiological conditions: we (i) imposed rapid ramp shortening on active muscle fibers to drive myosin detachment, (ii) abolished active force generation with a small molecule inhibitor, and (iii) stretched the muscle fibers to remove the overlap between the thick and thin filaments. The results lead to a four-state model that describes the sequence of structural changes in troponin and the thin filament during muscle activation.     

Morphometrical analysis of skeletal muscle fibre ageing and the effect of hypophysectomy and food restriction in the rat

Morphometrical analyses were carried out on muscle fibres from cross-sectional areas of the medial head of gastrocnemius muscles from 5 groups each of 4 male Wistar rats representing young (aged approximately 100 days), medium-aged (400 days), old (1,000 days), old hypophysectomized (1,000 days) and old food-restricted (1,000 days) rats. Approximately 18,000 muscle fibres were measured in each group. Rats were hypophysectomized when young at 60-70 days and food restriction began at the same age. The results show that there is a reduction in type 2 fibre size (measured as the mean equivalent circle diameter) from 59.7 micron in medium-aged controls to 50.3 micron in old controls, but there is no significant difference between the sizes of type 2 fibres in controls, hypophysectomized or food-restricted rats in old age. Analysis of type 1 fibres demonstrated no differences between young, medium-aged and old controls and the old food-restricted group, but type 1 fibres in the old hypophysectomized group (45.5 micron) were significantly smaller than in the old controls (54.6 micron) and old food-restricted rats (58.5 micron). In old controls there is a 4-fold increase in the range of fibre sizes (coefficient of variation) for type 1 fibres and a 2-fold increase for type 2 fibres, indicating fibre atrophy and hypertrophy. Long-term hypophysectomy is more effective than food restriction in preserving the smaller range of the coefficient of variation of fibre size seen in young controls.     

Reasons for the degeneration of ageing skeletal muscle: a central role for IGF-1 signalling

This paper examines two major possibilities for the striking loss of skeletal muscle mass and strength that occurs in very old animals and humans. It is concluded that muscle regeneration is not significantly impaired with age. Instead, it seems that individual myofibres undergo atrophy, with selective death of the fast type 2B myofibres, due to the combined effects of many age-related changes the main causes being: nutrition(under-nutrition and lack of vitamin D),decreased hormone levels (e.g growth hormone, testosterone), reduced physical activity, and a loss of nerves that innervate the muscles. The discussion focusses on the central role of a local muscle form of insulin-like growth factor-I (IGF-I) in muscle hypertrophy, atrophy and motor neurone loss. Reduced IGF-Isignalling is involved in muscle atrophy and results from decreased muscle exercise, reduced growth hormone and insulin levels, reduced vitamin D, and treatment with drugs like corticosteroids, dexamethasone, and cyclosporin. In addition, elevated levels of inflammatory cytokines like TNF-α and IL-6can cause muscle wasting (cachexia) although this is usually associated with disease, andTNF-α may also act (at least in part) by inhibiting IGF-I signalling. The possible clinical prevention of age-related muscle wasting (and associated motor neurone loss) by the locally acting muscle isoform of IGF-I is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Advanced glycation end-product accumulation and associated protein modification in type II skeletal muscle with aging

One mechanism that may influence the quality of skeletal muscle proteins, and explain the age-related decline in contractility, is protein damage. Advanced glycation end-products (AGE) in vivo are useful biomarkers of damage. In this study, comparison of extensor digitorum longus (EDL) muscles from young (8 months), old (33 months), and very old (36 months) Fischer 344 Brown Norway F1 (F344BNF1) hybrid rats shows that muscles from the very old rats have a significantly higher percentage of myofibers that immunolabel intracellularly for AGE-antibody 6D12 compared to the younger age group. The AGE-modified proteins, determined in the semimembranosus muscles from young (9 months) and old (27 months) F344 rats, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry include creatine kinase, carbonic anhydrase III, beta-enolase, actin, and voltage-dependent anion-selective channel 1. Moreover, there is a significant increase in AGE modification of beta-enolase with age. These results identify a common subset of proteins that contain AGE and suggest that metabolic proteins are targets for glycation with aging.     

Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis.

Sepsis was produced in rats by implanting into their abdominal cavities fecal pellets containing Escherichia coli (10(2) colony-forming units [CFU]) and Bacteroides fragilis (10(4) CFU). Control rats were implanted with sterile pellets. A febrile response and hyperlactacidemia marked the onset of the septic injury. Control and septic rats were killed 24 and 48 hr after implantations, and posterior leg muscles were removed. Muscles were homogenized to prepare soluble fractions containing calcium-independent lysosomal (cathepsins B and L) and calcium-dependent cytosolic (calpain) proteases. Cathepsin and calpain activities were then assayed using standard procedures. There were no alterations in cathepsins B or L activities during sepsis. Calpain activity in septic muscle was significantly higher than that in control muscles. In vitro calpain sensitivity to Ca2+ was also higher in septic muscle than in controls. The cysteine protease inhibitor leupeptin caused a quantitatively greater inhibition of calpain activity in septic than in control muscles. These data indicate that whereas sepsis has no effect on Ca(2+)-insensitive lysosomal proteases, it is associated with an elevation of the Ca(2+)-dependent cytosolic protease activity.     

Quantitative model for predicting lymph formation and muscle compressibility in skeletal muscle during contraction and stretch

Skeletal muscle is widely perceived as nearly incompressible despite the fact that blood and lymphatic vessels within the endomysial and perimysial spaces undergo significant changes in diameter and length during stretch and contraction. These fluid shifts between fascicle and interstitial compartments have proved extremely difficult to measure. In this paper, we propose a theoretical framework based on a space-filling hexagonal fascicle array to provide predictions of the displacement of blood and lymph into and out of the muscle's endomysium and perimysium during stretch and contraction. We also use this model to quantify the distribution of blood and initial lymphatic (IL) vessels within a fascicle and its perimysial space using data for the rat spinotrapezius muscle. On average, there are 11 muscle fibers, 0.4 arteriole/venule pairs, and 0.2 IL vessels per fascicle. The model predicts that the blood volume in the endomysial space increases 24% and decreases 22% for a 20% contraction and stretch, respectively. However, these significant changes in blood volume in the endomysium produce a change of only ～2% in fascicle cross-sectional area. In contrast, the entire muscle deviates from isovolumetry by 7% and 6% for a 20% contraction and stretch, respectively, largely attributable to the significantly larger blood volume changes that occur in the perimysial space. This suggests that arcade blood vessels in the perimysial space provide the primary pumping action required for the filling and emptying of ILs during muscular contraction and stretch.     

Microvascular adjustments during irreversible hemorrhagic shock in rat skeletal muscle

A study was made of microvessel response to hemorrhagic hypotension and the subsequent restoration of blood volume. The experiments were conducted on anesthetized rats in which the cremaster muscle was exteriorized for intravital microscopy. Variables measured during hypotension (40 mm Hg for 60 min) and after blood restoration (120 min observation) included systemic blood pressure, heart rate, arteriolar and venular diameter, sensitivity to epinephrine, velocity and volumetric flow rate. These findings were correlated with 24-hr survival statistics. The response to hemorrhagic hypotension is a reflection of two separate adjustments, cardiac output and peripheral vascular behavior. In survivors, the microvascular sequelae following blood replacement was one of continuous improvement of muscle perfusion, whereas in nonsurvivors the picture was one of progressive deterioration. The consistent hallmark of irreversibility, arteriolar hyposensitivity, was associated with a continuous falling off in mean arterial pressure despite restoration of normal blood volume.