Cécile Coudy-Gandilhon

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age‐related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein‐7 (Pax7) or laminin‐2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age‐related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre‐associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age‐dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.

Skeletal Muscle Lipid Content and Oxidative Activity in Relation to Muscle Fiber Type in Aging and Metabolic Syndrome

One of the most noticeable effects of aging is the reduction in skeletal muscle mass and strength (sarcopenia). The metabolic syndrome (MS) is also prevalent in old subjects, but its relevance to skeletal muscle characteristics has poorly been investigated. Immunohistochemical studies were performed with muscle biopsies from young (22 years) and old (73 years) men with and without MS to reveal age-dependent and MS-associated modifications of fiber-type characteristics. Atrophy of type II fibers and altered fiber shape characterized muscle aging in lean healthy men. In contrast, increased cross-sectional area of the most abundant type I and type IIA fibers, and reduced cytochrome c oxidase content in all fiber types, characterized MS. Aging and particularly MS were associated with accumulation of intramyocellular lipid droplets. Although lipids mostly accumulated in type I fibers, matrix-assisted laser desorption/ionization-mass spectrometry imaging of intramyocellular lipids did not distinguish fiber types, but clearly separated young, old, and MS subjects. In conclusion, our study suggests that MS in the elderly persons is associated with alterations in skeletal muscle at a fiber-type specific level. Overall, these fiber type-specific modifications may be important both for the age-related loss of muscle mass and strength and for the increased prevalence of MS in elderly subjects.

Proteomics of muscle chronological ageing in post-menopausal women

BackgroundMuscle ageing contributes to both loss of functional autonomy and increased morbidity. Muscle atrophy accelerates after 50 years of age, but the mechanisms involved are complex and likely result from the alteration of a variety of interrelated functions. In order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age.ResultsMuscle samples were compared between adult (56 years) and old (78 years) post-menopausal women. In addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. Two-dimensional gel electrophoreses with overlapping IPGs were used to improve the separation of muscle proteins. Overall, 1919 protein spots were matched between all individuals, 95 were differentially expressed and identified by mass spectrometry, and they corresponded to 67 different proteins. Our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. A fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton (myosin light-chains, troponin T, ankyrin repeat domain-containing protein-2, vinculin, four and a half LIM domain protein-3), which may account for alterations in contractile properties. In line with muscle contraction, we also identified proteins related to calcium signal transduction (calsequestrin-1, sarcalumenin, myozenin-1, annexins). Muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection (catalase, peroxiredoxins), ion homeostasis (carbonic anhydrases, selenium-binding protein 1) and detoxification (aldo-keto reductases, aldehyde dehydrogenases). Notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis (valosin-containing protein, proteasome subunit beta type-4, mitochondrial elongation factor-Tu).ConclusionsThis study describes the most extensive proteomic analysis of muscle ageing in humans, and identified 34 new potential biomarkers. None of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans.

Lower skeletal muscle capillarization in hypertensive elderly men

Aging strongly affects the skeletal muscle and is associated with microvascular dysfunctions. Age is also a primary risk factor for the metabolic syndrome, which is a cluster of metabolic and cardiovascular symptoms. Among the metabolic syndrome components, hypertension is the most prevalent in elderly subjects and has a central role in vascular alterations. Despite critical clinical outcomes, the effects of hypertension and metabolic syndrome on skeletal muscle capillarization have poorly been investigated during aging. In the present study, muscle biopsies from normotensive young (YO) and elderly (ELc) men, and elderly men with hypertension (EL-HT) or metabolic syndrome (EL-MS) were assessed for the number of capillaries around a fiber (CAF), capillary-to-fiber perimeter exchange (CFPE), length of contact to perimeter of fiber ratio (LC/PF), capillary tortuosity, and for extracellular matrix (ECM) embedding capillaries. As capillarization and muscle mitochondrial oxidative capacity may be associated, we also investigated cytochrome c oxidase (COX) content. Our findings indicate that capillarization and COX did not change between normotensive adult and old individuals. They further reveal that hypertension in elderly men is associated with reduced CAF (ELc: 5.2 ± 0.4, EL-HT: 4.1 ± 0.2, P<0.02 for type I fibers; ELc: 4.1 ± 0.2, EL-HT: 3.1 ± 0.3, P<0.03 for type IIA fibers), CFPE (ELc: 7.9 ± 0.7, EL-HT: 6.4 ± 0.4 capillaries/1000 μm, P<0.03 for type I fibers; ELc: 6.5 ± 0.4, EL-HT: 5.2 ± 0.4 capillaries/1000 μm, P<0.03 for type IIA fibers), LC/PF (ELc: 23.3 ± 1.2, EL-HT: 17.8 ± 0.6%, P<0.01 for type I fibers; ELc: 19.8 ± 1.1, EL-HT: 15.6 ± 0.8%, P<0.01 for type IIA fibers) and capillary tortuosity, and with ECM endomysium fibrosis. Capillary rarefaction also correlated with lower COX content in the old hypertensive muscle. No further modification occurred with metabolic syndrome in elderly men. Collectively, our results suggest that hypertension plays a central role in muscle capillarization during aging, and that the other components of metabolic syndrome do not make major additional changes in the aged skeletal muscle capillary network.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

Muscle loss associated changes of oxylipin signatures during biological aging: an exploratory study from the PROOF cohort

Characterizations of the multiple mechanisms determining biological aging are required to better understand the etiology and identify early biomarkers of sarcopenia. Oxylipins refer to a large family of signaling lipids involved in the regulation of various biological processes that become dysregulated during aging. To investigate whether comprehensive oxylipin profiling could provide an integrated and fine characterization of the early phases of sarcopenia, we performed a quantitative targeted metabolomics of oxylipins in plasma of 81-year old subjects from the PROOF cohort with decreased (n = 12), stable (n = 16) or increased appendicular muscle mass (n = 14). Multivariate and univariate analyses identified significant and concordant changes of oxylipin profiles according to the muscle status. Of note, 90 % of the most discriminant oxylipins were derived from EPA and DHA and were increased in the sarcopenic subjects. The oxylipins signatures of sarcopenic subjects revealed subtle activation of inflammatory resolution pathways, coagulation processes and oxidative stress as well as the inhibition of angiogenesis. Heat maps highlighted relationships between oxylipins and the cardiometabolic health parameters which were mainly lost in sarcopenic subjects. This exploratory study supports that targeted metabolomics of oxylipins could provide relevant and subtle characterization of early disturbances associated with muscle-loss during aging.

Increased Serpina3n release into circulation during glucocorticoid-mediated muscle atrophy: Serpina3n secretion in muscle atrophy

Glucocorticoids (GC) play a major role in muscle atrophy. As skeletal muscle is a secretory organ, characterization of the muscle secretome elicited by muscle atrophy should allow to better understand the cellular mechanisms and to identify circulating biomarkers of this condition. Our project aimed to identify the changes in the muscle secretome associated with GC‐induced muscle atrophy and susceptible to translate into circulation.