Intra-individual variation in body temperature and pectoral muscle size in nestling Alpine swifts Apus melba in response to an episode of inclement weather

The Alpine swift (Apus melba) forages on insects caught exclusively on the wing, implying that dependent nestlings face acute food shortage in periods of cold and rainy weather. Therefore, there should be strong selection on nestling swifts to evolve physiological strategies to cope with periods of undernutrition. We have investigated intra-individual changes in nestling pectoral muscle and body temperature in response to a 1-week period of inclement weather. The pectoral muscle is the largest reserves of proteins, and nestlings have to devote a large amount of energy in the maintenance of body temperature. The results show that nestling pectoral muscle size and body temperature were significantly reduced during the episode of inclement weather. Assuming that these physiological changes are adaptive, our study suggests that nestling swifts spare energy by a pronounced reduction (up to 18°C) in body temperature and use proteins from the pectoral muscle as a source of extra energy to survive prolonged periods of fasting.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

骨骼肌中肌浆网/内质网钙ATP酶的功能及其作用机制

骨骼肌是机体生命活动和能量代谢的重要场所,其代谢紊乱会诱发一系列肌肉疾病。Ca²⁺作为肌肉收缩过程的重要调节器,在骨骼肌的功能行使中发挥重要作用。骨骼肌细胞中Ca²⁺浓度主要受肌浆网/内质网钙ATP酶(sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase, SERCA)的调节。SERCA利用ATP水解产生的能量介导胞质Ca²⁺进入肌浆网内腔,维持胞质Ca²⁺平衡。SERCA功能的失调会引发一系列骨骼肌疾病,而SERCA活性受部分肌浆网蛋白质的调控,跨膜蛋白质PLN、SLN、MRLN、DWORF和sAnk1以及胞质蛋白质THADA和SAR,其通过磷酸化,进而调控SERCA的功能。本文对骨骼肌中SERCA的功能、调控SERCA的相关功能蛋白质的结构及其作用机制进行了总结,以期为骨骼肌相关疾病的治疗提供最新的思路和方法。     

Time-dependent changes in protein expression in rainbow trout muscle following hypoxia

Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia.     

Whole body proteome response to a dietary lysine imbalance in zebrafish Danio rerio

Lysine (Lys) is an indispensable amino acid (AA) and is generally the first limiting AA in most vegetable proteins used in fish feeds. Lys availability may thus limit protein synthesis and accretion, and growth of fish. Metabolic effects of dietary Lys imbalance were examined by 2D-proteomics using zebrafish as model. The Control diet (Lys: 2.47 g kg(-1)) was based on zebrafish carcass AA profiles previously obtained. Two other experimental diets were deficient in Lys [Lys(-); 1.34 g kg(-1)] and Lys added in excess [Lys(+); 4.63 g kg(-1)]. Fish growth was monitored from 33 to 49 days post-fertilization and the whole body proteome screened by means of two-dimension gel electrophoresis and mass spectrometry. Growth rate was negatively affected in group Lys(-). Comparative proteomic analysis showed 45 spots differentially expressed among groups. Twenty-nine of these proteins were identified revealing proteins involved in muscle growth, energy and lipid metabolism, eye lens differentiation, chaperone activity and apoptosis. Lys deficiency is accompanied by a down-regulation of muscle proteins and up-regulation of proteins affected by fasting, energy deficit, growth arrest and apoptosis. Excess Lys was accompanied by an up-regulation of proteins related to glycolysis, steroidogenesis and sexual maturation.     

Absence of aquaporin-4 in skeletal muscle alters proteins involved in bioenergetic pathways and calcium handling

Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4(-/-) compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4-/- muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca(2+) handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology.     

Changes in enzymes associated with energy metabolism during the early post mortem period in longissimus thoracis bovine muscle analyzed by proteomics

Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADP-dependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle.     

Proteomic investigation of changes in rat skeletal muscle after exercise-induced fatigue

The mechanisms of exercise-induced fatigue have not been investigated using proteomic techniques, an approach that could improve our understanding and generate novel information regarding the effects of exercise. In this study, the proteom alterations of rat skeletal muscle were investigated during exercise-induced fatigue. The proteins were extracted from the skeletal muscle of SD rat thigh, and then analyzed by two-dimensional electrophoresis and PDQuest software. Compared to control samples, 10 significantly altered proteins were found in exercise samples, two of them were upregulated and eight of them were downregulated. These proteins were identified by MALDI TOF-MS. The two upregulated proteins were identified as MLC1 and myosin L2 (DTNB) regulatory light-chain precursors. The eight decreased proteins are Glyceraldehyde-3-phosphate Dehydrogenas (GAPDH); Beta enolase; Creatine kinase M chain (M-CK); ATP-AMP Transphosphorylase (AK1); myosin heavy chain (MHC); actin; Troponin I, fast-skeletal muscle (Troponin I fast-twitch isoform), fsTnI; Troponin T, fast-skeletal muscle isoforms (TnTF). In these proteins, four of the eight decreased proteins are related directly or indirectly to exercise induced fatigue. The other proteins represent diverse sets of proteins including enzymyes related to energy metabolism, skeletal muscle fabric protein and protein with unknown functions. They did not exhibit evident relationship with exercise-induced fatigue. Whereas the two identified increased proteins exhibit evident relationship with fatigue. These findings will help in understanding the mechanisms involved in exercise-induced fatigue.     

Metabolism of 3-methyl histidine in experimentally induced protein-energy malnutrition in rats: Urinary excretion and muscle content of 3-methyl histidine

Urinary excretion and muscle content of 3-Methyl Histidine was determined, in rats subjected to protein or energy depletion for a period of 7–8 weeks. Urinary excretion of 3-Methyl Histidine was significantly reduced both in absolute amounts and in relation to creatinine excretion in both the protein and energy depleted rats. The muscle content of 3-Methyl Histidine was reduced only in protein depleted group. After rehabilitation, the protein depleted rats excreted normal levels of 3-Methyl Histidine and their muscle 3-Methyl Histidine content also returned to normal levels. The results are suggestive of altered catabolic rates of muscle proteins in protein energy malnutrition.     

Quantitative proteome analysis of age‐related changes in mouse gastrocnemius muscle using mTRAQ

Aging is associated with a progressive loss of skeletal muscular function that often leads to progressive disability and loss of independence. Although muscle aging is well documented, the molecular mechanisms of this condition still remain unclear. To gain greater insight into the changes associated with aging of skeletal muscle, we performed quantitative proteomic analyses on young (6 months) and aged (27 months) mouse gastrocnemius muscles using mTRAQ stable isotope mass tags. We identified and quantified a total of 4585 peptides corresponding to 236 proteins (protein probability >0.9). Among them, 33 proteins were more than 1.5‐fold upregulated and 20 proteins were more than 1.5‐fold downregulated in aged muscle compared with young muscle. An ontological analysis revealed that differentially expressed proteins belonged to distinct functional groups, including ion homeostasis, energy metabolism, protein turnover, and Ca²⁺ signaling. Identified proteins included aralar1, β‐enolase, fatty acid‐binding protein 3, 3‐hydroxyacyl‐CoA dehydrogenase (Hadh), F‐box protein 22, F‐box, and leucine‐rich repeat protein 18, voltage‐dependent L‐type calcium channel subunit beta‐1, ryanodine receptor (RyR), and calsequestrin. Ectopic expression of calsequestrin in C2C12 myoblast resulted in decreased activity of nuclear factor of activated T‐cells and increased levels of atrogin‐1 and MuRF1 E3 ligase, suggesting that these differentially expressed proteins are involved in muscle aging.     

Cellular Localization and Associations of the Major Lipolytic Proteins in Human Skeletal Muscle at Rest and during Exercise

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis.     

Re-patterning of skeletal muscle energy metabolism by fat storage-inducing transmembrane protein 2

Triacylglyceride stored in cytosolic lipid droplets (LDs) constitutes a major energy reservoir in most eukaryotes. The regulated turnover of triacylglyceride in LDs provides fatty acids for mitochondrial β-oxidation and ATP generation in physiological states of high demand for energy. The mechanisms for the formation of LDs in conditions of energy excess are not entirely understood. Fat storage-inducing transmembrane protein 2 (FIT2/FITM2) is the anciently conserved member of the fat storage-inducing transmembrane family of proteins implicated to be important in the formation of LDs, but its role in energy metabolism has not been tested. Here, we report that expression of FIT2 in mouse skeletal muscle had profound effects on muscle energy metabolism. Mice with skeletal muscle-specific overexpression of FIT2 (CKF2) had significantly increased intramyocellular triacylglyceride and complete protection from high fat diet-induced weight gain due to increased energy expenditure. Mass spectrometry-based metabolite profiling suggested that CKF2 skeletal muscle had increased oxidation of branched chain amino acids but decreased oxidation of fatty acids. Glucose was primarily utilized in CKF2 muscle for synthesis of the glycerol backbone of triacylglyceride and not for glycogen production. CKF2 muscle was ATP-deficient and had activated AMP kinase. Together, these studies indicate that FIT2 expression in skeletal muscle plays an unexpected function in regulating muscle energy metabolism and indicates an important role for lipid droplet formation in this process.     

Proteomic analysis of rat laryngeal muscle following denervation

Laryngeal muscle atrophy induced by nerve injury is a major factor contributing to the disabling symptoms associated with laryngeal paralysis. Alterations of global proteins in rat laryngeal muscle following denervation were, therefore, studied using proteomic techniques. Twenty-eight adult Sprague-Dawley rats were divided into normal control and denervated groups. The thyroarytenoid (TA) muscle was excised 60 days after right recurrent laryngeal nerve was resected. Protein separation and identification were preformed using 2-DE and MALDI-MS with database search. Forty-four proteins were found to have significant alteration in expression level after denervation. The majority of these proteins (57%), most of them associated with energy metabolism, cellular proliferation and differentiation, signal transduction and stress reaction, were decreased levels of expression in denervated TA muscle. The remaining 43% of the proteins, most of them involved with protein degradation, immunoreactivity, injury repair, contraction, and microtubular formation, were found to have increased levels of expression. The protein modification sites by phosphorylation were detected in 22% of the identified proteins that presented multiple-spot patterns on 2-D gel. Significant changes in protein expression in denervated laryngeal muscle may provide potential therapeutic strategies for the treatment of laryngeal paralysis.     

Differential expression of metabolic genes essential for glucose and lipid metabolism in skeletal muscle from spinal cord injured subjects

Skeletal muscle plays an important role in the regulation of energy homeostasis; therefore, the ability of skeletal muscle to adapt and alter metabolic gene expression in response to changes in physiological demands is critical for energy balance. Individuals with cervical spinal cord lesions are characterized by tetraplegia, impaired thermoregulation, and altered skeletal muscle morphology. We characterized skeletal muscle metabolic gene expression patterns, as well as protein content, in these individuals to assess the impact of spinal cord injury on critical determinants of skeletal muscle metabolism. Our results demonstrate that mRNA levels and protein expression of skeletal muscle genes essential for glucose storage are reduced, whereas expression of glycolytic genes is reciprocally increased in individuals with spinal cord injury. Furthermore, expression of genes essential for lipid oxidation is coordinately reduced in spinal cord injured subjects, consistent with a marked reduction of mitochondrial proteins. Thus spinal cord injury resulted in a profound and tightly coordinated change in skeletal muscle metabolic gene expression program that is associated with the aberrant metabolic features of the tissue.     

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Proteomic identification of age-dependent protein nitration in rat skeletal muscle

Age-related protein nitration was studied in skeletal muscle of Fisher 344 and Fisher 344/Brown Norway (BN) F1 rats by a proteomic approach. Proteins from young (4 months) and old (24 months) Fisher 344 rats and young (6 months) and old (34 months) Fisher 344/BN F1 animals were separated by 2-D gel electrophoresis. Western blot showed an age-related increase in the nitration of a few specific proteins, which were identified by MALDI-TOF MS and ESI-MS/MS. We identified age-dependent apparent nitration of beta-enolase, alpha-fructose aldolase, and creatine kinase, which perform important functions in muscle energy metabolism, suggesting that the nitration of such key proteins can be, in part, responsible for the decline of muscle motor function of the muscle. Furthermore, we have identified the apparent nitration of succinate dehydrogenase, rab GDP dissociation inhibitor beta (GdI-2), triosephosphate isomerase, troponin I, alpha-crystallin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).