Daniele Capitanio

Long term bed rest with and without vibration exercise countermeasures: Effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2‐D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (∼50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (∼35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ∼15% in cross‐sectional area and of ∼22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z‐disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl‐L‐carnitine supplementation

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow‐oxidative to fast‐glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl‐L‐carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2‐D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow‐twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal‐loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.

Molecular Signatures of Amyotrophic Lateral Sclerosis Disease Progression in Hind and Forelimb Muscles of an SOD1G93A Mouse Model

AIMS This study utilized proteomics, biochemical and enzymatic assays, and bioinformatics tools that characterize protein alterations in hindlimb (gastrocnemius) and forelimb (triceps) muscles in an amyotrophic lateral sclerosis (ALS) (SOD1(G93A)) mouse model. The aim of this study was to identify the key molecular signatures involved in disease progression. RESULTS Both muscle types have in common an early down-regulation of complex I. In the hindlimb, early increases in oxidative metabolism are associated with uncoupling of the respiratory chain, an imbalance of NADH/NAD(+), and an increase in reactive oxygen species (ROS) production. The NADH overflow due to complex I inactivation induces TCA flux perturbations, leading to citrate production, triggering fatty acid synthase (FAS), and lipid peroxidation. These early metabolic changes in the hindlimb followed by sustained and comparatively higher metabolic and cytoskeletal derangements over time precede and may catalyze the progressive muscle wasting in this muscle at the late stage. By contrast, in the forelimb, there is an early down-regulation of complexes I and II that is associated with the reduction of oxidative metabolism, which promotes metabolic homeostasis that is accompanied by a greater cytoskeletal stabilization response. However, these early compensatory systems diminish by a later time point. INNOVATION The identification of potential early- and late-stage disease molecular signatures in an ALS model: muscle albumin, complex I, complex II, citrate synthase, FAS, and phosphoinositide 3-kinase functions as diagnostic markers and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), Sema-3A, and Rho-associated protein kinase 1 (ROCK1) play the role of disease progression markers. CONCLUSION The differing pattern of cellular metabolism and cytoskeletal derangements in the hind and forelimb identifies the potential dysmetabolism/hypermetabolism molecular signatures associated with disease progression, which may serve as diagnostic/disease progression markers in ALS patients.

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996 ). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Δtpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutants impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Δtpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Δtpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Δtpi1 mutant may occur in relation to the fraction of ethanol present in the media. Copyright

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF‐1α), energy balance (AMPK), mitochondrial biogenesis (PGC‐1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2‐D DIGE, mass spectrometry and antigen–antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF‐1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up‐regulation of adenosylhomocysteinase and acetyl‐CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC‐1α, a master regulator of mitochondrial biogenesis, was down‐regulated. CH was characterized by the up‐regulation of enzymes involved in antioxidant defense, in aldehyde bio‐product detoxification and in misfolded protein degradation. In addition, a general down‐regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro‐apoptotic molecules increased accompained by down‐regulation of specific target proteins.

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.

Muscle proteomics reveals novel insights into the pathophysiological mechanisms of collagen vi myopathies

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Changes in Muscle Cell Metabolism and Mechanotransduction Are Associated with Myopathic Phenotype in a Mouse Model of Collagen VI Deficiency

This study identifies metabolic and protein phenotypic alterations in gastrocnemius, tibialis anterior and diaphragm muscles of Col6a1−/− mice, a model of human collagen VI myopathies. All three muscles of Col6a1−/− mice show some common changes in proteins involved in metabolism, resulting in decreased glycolysis and in changes of the TCA cycle fluxes. These changes lead to a different fate of α-ketoglutarate, with production of anabolic substrates in gastrocnemius and tibialis anterior, and with lipotoxicity in diaphragm. The metabolic changes are associated with changes of proteins involved in mechanotransduction at the myotendineous junction/costameric/sarcomeric level (TN-C, FAK, ROCK1, troponin I fast) and in energy metabolism (aldolase, enolase 3, triose phosphate isomerase, creatine kinase, adenylate kinase 1, parvalbumin, IDH1 and FASN). Together, these change may explain Ca2+ deregulation, impaired force development, increased muscle-relaxation-time and fiber damage found in the mouse model as well as in patients. The severity of these changes differs in the three muscles (gastrocnemius

Changes in muscle proteomics in the course of the Caudwell Research Expedition to Mt. Everest.

This study employed differential proteomic and immunoassay techniques to elucidate the biochemical mechanisms utilized by human muscle (vastus lateralis) in response to high altitude hypoxia exposure. Two groups of subjects, participating in a medical research expedition (A, n = 5, 19d at 5300 m altitude; B, n = 6, 66d up to 8848 m) underwent a ≈ 30% drop of muscular creatine kinase and of glycolytic enzymes abundance. Protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced both in A and, particularly, in B. Restriction of α‐ketoglutarate toward succinyl‐CoA resulted in increased prolyl hydroxylase 2 and glutamine synthetase. Both A and B were characterized by a reduction of elongation factor 2alpha, controlling protein translation, and by an increase of heat shock cognate 71 kDa protein involved in chaperone‐mediated autophagy. Increased protein levels of catalase and biliverdin reductase occurred in A alongside a decrement of voltage‐dependent anion channels 1 and 2 and of myosin‐binding protein C, suggesting damage to the sarcomeric structures. This study suggests that during acclimatization to hypobaric hypoxia the muscle behaves as a producer of substrates activating a metabolic reprogramming able to support anaplerotically the tricarboxylic acid cycle, to control protein translation, to prevent energy expenditure and to activate chaperone‐mediated autophagy.

Specific protein changes contribute to the differential muscle mass loss during ageing

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D‐DIGE, MALDI‐TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3‐ and 22‐month‐old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative‐stress‐induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine‐residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.