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O P E N A C C E S S S O U R C E : American Journal of Physiology - Endocrinology and Metabolism
Abstract
The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated β-galactosidase (SA-β-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-β-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (−70%, P = 0.011) and tended to have lower TNF-α mRNA levels (−40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.
INTRODUCTION
The loss of muscle functions, including degenerative loss of strength and mass, is a well-known feature of aging in human, which contributes to the progressive loss of autonomy in the elderly (25, 44). Despite the large number of studies indicating a protective role of physical activity against the sequels of aging (37), the molecular mechanisms by which exercise counteracts skeletal muscle aging need to be further investigated.
Telomeres are specialized nucleoprotein (TTAGGG repeats) caps at the end of the chromosomes, which protect genome stability (2). As each cell division results in telomere attrition, aging induces telomere shortening (7). Loss of telomere length impairs tissue renewal (7, 27). Adult skeletal muscle is considered as a postmitotic tissue (22). Therefore, the capacity to regenerate new myonuclei either for fiber growth or for fiber repair lies in one specific mononucleated precursor cells: the satellite cells (29). Besides the well-established loss of telomeric DNA related to the replicative history of somatic cells, telomere erosion is not constant and differs between people. These variations can be explained by environmental conditions such as oxidative stress and inflammation (38). Chronic low-grade systemic inflammation is a common manifestation of aging, with higher circulating levels of proinflammatory cytokines such as TNF-α (8). While chronic inflammation is higher with sedentary behavior (11), studies have evidenced that endurance training may reduce markers of systemic and skeletal muscle inflammation (5, 26, 46). Endurance training may have beneficial effects on telomeres. However, more research is needed to establish a consensus (3). For example, long-term high-intensity cross-country skiing provided a protective effect on muscle telomere length in older athletes compared with subjects exercising at a moderate level of activity (31), while middle age experienced runners showed shorter skeletal muscle telomeres compared with age-matching sedentary individuals (35). Moreover, the impact of endurance training on telomere integrity in a specific cell population such as satellite cells remains largely unknown.
Cellular senescence is a durable cell cycle arrest resulting notably in the accumulation of cyclin-dependent kinase inhibitor 2A (p16) and/or cyclin-dependent kinase inhibitor 1 (p21) (12, 21, 36). Telomere damage can be visualized by immunofluorescence microscopy using antibodies against DNA damage response factors, such as tumor suppressor p53-binding protein 1 (53BP1), coupled with telomere detection by fluorescent in situ hybridization (FISH) or immunofluorescence (14, 32, 41). A cell becomes senescent when multiple telomeres in the nucleus display DNA damage markers, turning them into so-called telomere dysfunction-induced foci (TIF) (41). Senescence can be induced by many stimuli (10). In addition to senescence conveyed by repeated cell divisions (i.e., replicative senescence or telomere-dependent senescence), stress, such as oxidative stress, can also induce cellular senescence in a telomere-independent manner and lead to increased p16 expression (9, 10, 43). This type of growth arrest is called stress or aberrant signaling-induced senescence (16). Higher lysosomal β-galactosidase activity is often reported in senescent cells, leading to measurable senescence-associated β-galactosidase (SA-β-Gal) activity (21). Moreover, senescent cells induce a cellular program called senescence-associated secretory phenotype (SASP), resulting in the secretion of cytokines with potent proinflammatory effects (20, 21). Indeed, SASP involves the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance, notably by recruiting macrophages (20, 21). SASP cytokine production depends largely on stress-induced nuclear factor-κB (NF-κB) signaling, which promotes the transcription of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) coding genes (12).
The aim of the present study was to determine if a better endurance training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle of older compared with younger subjects, with a specific focus on satellite cells.
MATERIALS AND METHODS
Human study.
The present work has been conducted in parallel with another one looking at the effects of aging and the training status on mitophagy on the same muscle samples (4). Briefly, 34 healthy men were recruited, and their cardiorespiratory fitness was assessed during a maximal incremental fitness test on a cycle ergometer (Cyclus III; RBM Electronics). Based on their age and physical level, the subjects were randomly divided into four groups: young sedentary (YS) (n = 9), young trained (YT) (n = 9), old sedentary (OS) (n = 8), and old trained (OT) (n = 8). None of the sedentary subjects were engaged in any weekly physical activity session for at least 5 yr whereas physically active subjects were all experienced cyclists and reported ≥6 h training a week for at least 5 yr. None of the volunteers participated in resistance training before their enrollment in our study. One week later, a skeletal muscle biopsy was taken in the fasted state from the vastus lateralis muscle under local anesthesia with 1 ml of Xylocaine 2% (AstraZeneca) using a Bergström needle. A biopsy of ~70–100 mg of skeletal muscle was immediately frozen in liquid nitrogen for biomolecular analyses and satellite cell isolation while ~20 mg were embedded in optimum cutting temperature compound and immediately frozen in cooled isopentane so immunofluorescence could be performed on muscle sections. Before muscle biopsy, all subjects were asked to refrain from strenuous physical activity for 2 days and from alcohol for 1 day. The protocol was approved by the local Ethical Committee of the Université Catholique de Louvain and conducted in accordance with the Declaration of Helsinki. All participants provided their written consent after being fully informed about the experimental procedure.
RNA extraction and quantitative real-time PCR.
About 30 mg of muscle biopsy were homogenized using a Polytron (Kinematica) in 1 ml Trizol reagent (Invitrogen). RNA isolation was achieved according to the manufacturer’s instructions. RNA quality and quantity were assessed by Nanodrop (Thermo Fisher Scientific) spectrophotometry. Reverse transcription was performed from 1 µg RNA using the iScriptcDNA Synthesis Kit (Bio-Rad), following the manufacturer’s instructions. PCR analyzes were conducted using the following conditions: 3 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C using CFX Connect Real-Time System (Bio-Rad Laboratories). All samples were run in duplicate. Each reaction was processed in a 10-µl volume containing 4.8 µl SsoAdvanced Universal SYBR Green SuperMix (Bio-Rad), 0.1 µl of each primer (100 nM final), and 5 µl cDNA at the appropriate dilution. Melting curves were systematically performed to ensure the quality of the analysis. All mRNA levels were normalized to β2-microglobulin (β2M). Primers sequences are provided in Table 1.
Table 1. Primers sequences
Immunofluorescence on cryosections.
Cryosections (6 µm) were air dried and directly blocked with PBS/20% normal goat serum for 30 min at room temperature. Slides were then incubated for 1 h at 37°C with the following primary antibodies: anti-laminin (1/200, NB600-883, Novus) and anti-CD68 (1/200, no. 76437, Cell Signaling). After several washes with PBS, slides were incubated for 1 h at room temperature with the following secondary antibodies: Alexa Fluor 568 anti-mouse (A11031) and Alexa Fluor 488 anti-rabbit (A25034) (1/500, Thermo Fisher Scientific). Samples were mounted with Vectashield containing DAPI (Vector Laboratories). Images were acquired with a Vert.A1 (Zeiss) microscope using a ×40 objective.
Satellite cell isolation.
Satellite cell isolation was performed on four biopsies per group. About 20 mg of muscle were scissor minced, and the tissue fragments were plated onto collagen-coated dishes. The explants were maintained in the dish with a thin layer of Matrigel (Corning Life Sciences) and incubated in DMEM (Dutscher) supplemented with 10% FBS (VWR) and 1% Ultroser G (Pall Biosepra) at 37°C in humidified air with 5% CO2. After 6–8 days, cells migrated out of the explants and were enzymatically harvested using dispase (Merck). Satellite cells were purified using an immunomagnetic bead-based sorting system with high gradient magnetic cell separation (MACS) microbeads (Miltenyi Biotec) directly linked to an antibody against CD56 (BD559044, BD Biosciences).
Senescence-associated galactosidase staining in cultured satellite cells.
Cultured satellite cells were fixed with 3.7% paraformaldehyde (PFA) (Merck) for 5 min at room temperature before incubation at 37°C in freshly prepared SA-β-Gal staining solution [1 mg/ml 5-bromo-4-chloro-3-indoly β-d-galactoside (X-Gal) (Merck), 40 mM citric acid/sodium phosphate (Merck), pH 5.5, 5 mM potassium ferrocyanide (Merck), 5 mM potassium ferricyanide, 150 mM NaCl (Merck), and 2 mM MgCl2 (Merck)]. Blue cells were counted as senescent cells. Two hundred cells were counted for each subject.
TIF detection and telomere length analysis in satellite cells.
As previously reported (15), TIF detection was performed to evaluate telomere integrity. After fixation with 3.7% PFA (Merck) for 15 min and permeabilization [20 mM Tris·HCl (Merck), pH 8.0, 50 mM NaCl (Merck), 3 mM MgCl2 (Merck), 300 mM sucrose (Merck), and 0.5% Triton X-100 (Merck)], the antibody 53BP1 (NB100-304, Novus) was used to visualized DNA damage by immunofluorescence microscopy while telomeres were marked by fluorescent in situ hybridization (FISH). Colocalization events between 53BP1 and telomeric DNA are counted as TIF. Briefly, after immunofluorescence against 53BP1, satellite cells were fixed again with 3.7% PFA for 2 min and treated with ribonuclease (100 ug/ml) for 1 h at 37°C. After a second incubation with permeabilization buffer (20 mM Tris·HCl, pH 8.0, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100) for 10 min at room temperature and refixation for 2 min with 3.7% PFA, cells were dehydrated through successive baths of 70, 80, 90, and 100% ethanol for 2 min each. Telomeres were hybridized with 50 nM TeloG Exiqon Locked Nucleic Acid (LNA) red probe GGGTtAGGGttAGgGTTAGGGttAGGGttAGGGtTA (TAMRA) (small letters indicate LNA-modified bases) for 2 h at room temperature after denaturation for 3 min at 85°C. After several washes, samples were mounted with Vectashield containing DAPI (Vector Laboratories). Telomere length in each culture of satellite cells was normalized to the signal obtained in an immortalized mouse myoblast cell line (C2C12, ATCC) seeded on the same slide. Images were acquired with a Cell Observer spinning disk (Zeiss) confocal microscope using a ×100 objective and analyzed using ImageJ software (National Institutes of Health).
Statistics.
All results are presented as means ± SE. Statistical analyses were performed using GraphPad Prism 7.0. Data were analyzed by a two-way ANOVA, and when a main effect was found, Bonferroni post hoc tests were performed. Statistical significance was set at P ≤ 0.05.
RESULTS
Subjects characteristics.
Subjects characteristics can be found in (4). Briefly, the mean age of the young subjects (YS + YT) was 22 ± 1 yr and the mean age of the old subjects (OS + OT) was 67 ± 1 yr, with no difference between the sedentary and trained groups. Body mass index (BMI) was higher in OS (26.4 ± 1.4 kg/m2) than YS (23.0 ± 1.1 kg/m2, P = 0.039) and YT (23.1 ± 1.0 kg/m2, P = 0.049). V̇o2max was ~25% higher in YT compared with YS (61 ± 3 vs. 48 ± 4 ml·min−1·kg−1, P = 0.035) and ~50% higher in OT compared with OS (44 ± 4 vs. 30 ± 2 ml·min−1·kg−1, P = 0.029). OT had similar V̇o2max values as YS (~45 ml·min−1·kg−1). Wmax was 75% higher in OT compared with OS (3.3 ± 0.2 vs. 1.9 ± 0.2 W/kg, P < 0.001) and 40% higher in YT compared with YS (4.3 ± 0.3 vs. 3.0 ± 0.2 W/kg, P < 0.001).
Aging results in a higher number of dysfunctional telomeres in satellite cells without any effect of the training status.
Neither age nor the training status appeared to modify the relative telomere length in cultured satellites cells (Fig. 1, A and B). However, the evaluation of telomere length by FISH based on microscopy data analysis is likely not sensitive enough to detect small changes in telomere length. This is why we relied on another approach. We chose to evaluate the loss of telomere integrity, through the detection of TIF. Our data indicated that the percentage of cultured satellite cells containing three or more TIF per nucleus increased with aging (main effect of aging, P < 0.0001; Fig. 2, A and B). Post hoc analysis showed that OS and OT had a higher number of cells containing greater than or equal to three TIF per nucleus compared with either YS (+35%, P = 0.0007 and +36%, P = 0.0006, respectively) or YT (+39%, P = 0.0003 and +40%, P = 0.0003) (Fig. 2, A and B). The training status did not modify the percentage of cells containing greater than or equal to three TIF per nucleus.
Fig. 1.
Relative telomere length in cultured satellite cells. A: telomere length is expressed as arbitrary units. Results are normalized to C2C12 signal and reported to the young sedentary (YS) group who was arbitrarily set to a value of 1; A.U., arbitrary units. B: illustration of a quantitative fluorescence in situ hybridization (qFISH) signal. Nuclei are identified by DAPI in blue, and telomeres are marked by the qFISH probe in red. Human satellite cells showed a lower qFISH signal compared with the C2C12 immortalized cell line (marked by white arrows). Scale bar = 50 µm. Values are expressed as means ± SE; n = 4/group. YT, young trained; OS, old sedentary; OT, old trained.
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