Gymnastik- och idrottshögskolan, GIH

Age-related loss of muscle mass and function is underpinned by changes at the myocellular level. However, our understanding of the aged muscle phenotype might be confounded by factors secondary to ageing per se, such as inactivity and adiposity. Here, using healthy, lean, recreationally active, older men, we investigated the impact of ageing on myocellular properties in skeletal muscle. Muscle biopsies were obtained from young men (22 ± 3 years, n = 10) and older men (69 ± 3 years, n = 11) matched for health status, activity level and body mass index. Immunofluorescence was used to assess myofibre composition, morphology (size and shape), capillarization, the content of satellite cells and myonuclei, the spatial relationship between satellite cells and capillaries, denervation and myofibre grouping. Compared with young muscle, aged muscle contained 53% more type I myofibres, in addition to smaller (-32%) and misshapen (3%) type II myofibres (P < 0.05). Aged muscle manifested fewer capillaries (-29%) and satellite cells (-38%) surrounding type II myofibres (P < 0.05); however, the spatial relationship between these two remained intact. The proportion of denervated myofibres was ∼2.6-fold higher in old than young muscle (P < 0.05). Aged muscle had more grouped type I myofibres (∼18-fold), primarily driven by increased size of existing groups rather than increased group frequency (P < 0.05). Aged muscle displayed selective deterioration of type II myofibres alongside increased denervation and myofibre grouping. These data are key to understanding the cellular basis of age-related muscle decline and reveal a pressing need to fine-tune strategies to preserve type II myofibres and innervation status in ageing populations.

Background

Sarcopenia is thought to be underlined by age-associated anabolic resistance and dysregulation of intracellular signalling pathways. However, it is unclear whether these phenomena are driven by ageing per se or other confounding factors.

Methods

Lean and healthy young (n = 10, 22 ± 3 years, BMI; 23.4 ± 0.8 kg/m2) and old men (n = 10, 70 ± 3 years, BMI; 22.7 ± 1.3 kg/m2) performed unilateral resistance exercise followed by intake of essential amino acids (EAA). Muscle biopsies were collected from the rested and the exercised leg before, immediately after and 60 and 180 min after EAA intake. Muscle samples were analysed for amino acid concentrations, muscle protein synthesis (MPS) and associated anabolic signalling.

Results

Following exercise, peak plasma levels of EAA and leucine were similar between groups, but the area under the curve was ~11% and ~28% lower in Young (p < 0.01). Absolute levels of muscle EAA and leucine peaked 60 min after exercise, with ~15 and ~21% higher concentrations in the exercising leg (p < 0.01) but with no difference between groups. MPS increased in both the resting (~0.035%·h−1 to 0.056%·h−1, p < 0.05) and exercising leg (~0.035%·h−1 to 0.083%·h−1, p < 0.05) with no difference between groups. Phosphorylation of S6K1Thr389 increased to a similar extent in the exercising leg in both groups but was 2.8-fold higher in the resting leg of Old at the 60 min timepoint (p < 0.001). Phosphorylation of 4E-BP1Ser65 increased following EAA intake and exercise, but differences between legs were statistically different only at 180 min (p < 0.001). However, phosphorylation of this site was on average 78% greater across all timepoints in Old (p < 0.01). Phosphorylation of eEF2Thr56 was reduced (~66% and 39%) in the exercising leg at both timepoints after EAA intake and exercise, with no group differences (p < 0.05). However, phosphorylation at this site was reduced by ~27% also in the resting leg at 60 min, an effect that was only seen in Old (p < 0.01). Total levels of Rheb (~45%), LAT1 (~31%) and Rag B (~31%) were higher in Old (p < 0.001).

Conclusion

Lean and healthy old men do not manifest AR as evidenced by potent increases in MPS and mTORC1 signalling following EAA intake and exercise. Maintained anabolic sensitivity with age appears to be a function of a compensatory increase in basal levels of proteins involved in anabolic signalling. Therefore, our results suggest that age per se does not appear to cause AR in human skeletal muscle.

BACKGROUND: Exercising with low muscle glycogen content can improve training adaptation, but the mechanisms underlying the muscular adaptation are still largely unknown. In this study, we measured substrate utilization and cell signaling in different muscle fiber types during exercise and investigated a possible link between these variables.

METHODS: Five subjects performed a single leg cycling exercise in the evening (day 1) with the purpose of reducing glycogen stores. The following morning (day 2), they performed two-legged cycling at ∼70% of VO_2peak for 1 h. Muscle biopsies were taken from both legs pre- and post-exercise for enzymatic analyses of glycogen, metabolite concentrations using LC-MS/MS-based quantification, and protein signaling using Western blot in pools of type I or type II fibers.

RESULTS: Glycogen content was 60-65% lower for both fiber types (P < 0.01) in the leg that exercised on day 1 (low leg) compared to the other leg with normal level of glycogen (normal leg) before the cycling exercise on day 2. Glycogen utilization during exercise was significantly less in both fiber types in the low compared to the normal leg (P < 0.05). In the low leg, there was a 14- and 6-fold increase in long-chain fatty acids conjugated to carnitine in type I and type II fibers, respectively, post-exercise. This increase was 3-4 times larger than in the normal leg (P < 0.05). Post-exercise, mTOR^Ser2448 phosphorylation was increased in both fiber types in the normal leg (P < 0.05) but remained unchanged in both fiber types in the low leg together with an increase in eEF2^Thr56 phosphorylation in type I fibers (P < 0.01). Exercise induced a reduction in the autophagy marker LC3B-II in both fiber types and legs, but the post-exercise level was higher in both fiber types in the low leg (P < 0.05). Accordingly, the LC3B-II/I ratio decreased only in the normal leg (75% for type I and 87% for type II, P < 0.01).

CONCLUSIONS: Starting an endurance exercise session with low glycogen availability leads to profound changes in substrate utilization in both type I and type II fibers. This may reduce the mTORC1 signaling response, primarily in type I muscle fibers, and attenuate the normally observed reduction in autophagy.

Muscle disuse has rapid and debilitating effects on muscle mass and overall health, making it an important issue from both scientific and clinical perspectives. However, the myocellular adaptations to muscle disuse are not yet fully understood, particularly those related to the myonuclear permanence hypothesis. Therefore, in this study, we assessed fiber size, number of myonuclei, satellite cells, and capillaries in human gastrocnemius muscle after a period of immobilization following an Achilles tendon rupture. Six physically active patients (5M/1F, 43 {plus minus} 15 years) were recruited to participate after sustaining an acute unilateral Achilles tendon rupture. Muscle biopsies were obtained from the lateral part of the gastrocnemius before and after six weeks of immobilization using a plaster cast and orthosis. Muscle fiber characteristics were analyzed in tissue cross-sections and isolated single fibers using immunofluorescence and high-resolution microscopy. Immobilization did not change muscle fiber type composition nor cross-sectional area of type I or type II fibers, but muscle fiber volume tended to decline by 13% (p=0.077). After immobilization, the volume per myonucleus was significantly reduced by 20% (p=0.008). Myonuclei were not lost in response to immobilization but tended to increase in single fibers and type II fibers. No significant changes were observed for satellite cells or capillaries. Myonuclei were not lost in the gastrocnemius muscle after a prolonged period of immobilization, which may provide support to the myonuclear permanence hypothesis in human muscle. Capillaries remained stable throughout the immobilization period, whereas the response was variable for satellite cells, particularly in type II fibers.

Maintaining muscle mass is crucial for health and physical activity. Around age 40, muscle mass begins to decline, potentially leading to sarcopenia, a condition associated with frailty and increased fall risk. Age-related muscle loss is complex and multifactorial. The prevailing view is that this loss is driven by anabolic resistance, which is a reduced capacity to increase muscle protein synthesis (MPS) after anabolic cues, i.e., essential amino acids (EAA) or resistance exercise (REx). Mechanistically, this is thought to be underpinned by dysregulation of the mTORC1 signaling pathway. However, it is unclear whether anabolic resistance contributes to muscle loss in healthy, physically active older adults or if studies supporting this have been confounded by other factors, e.g., inactivity and adiposity. Aging also induces changes at the myocellular level, such as satellite cell loss and morphological alterations, but whether these changes are due to aging itself or lifestyle factors is still being debated.           

This thesis examined how anabolic cues impact MPS, mTORC1 signaling, and markers of protein degradation in young and older men. Emphasis was on performing analyses on whole muscle samples and in type I and type II fibers separately. Further aims were to investigate features of muscle fibers in young and older men, focusing on morphology, satellite cells, capillarization, and denervation-reinnervation cycles. The final aim was to develop a valid and fast method for fiber type identification of isolated fibers.           

In paper I, the MPS and mTORC1 signaling response was examined in young and older men after EAA intake alone and combined with REx. The results showed comparable rates of MPS across age groups in response to EAA intake, both alone and with REx. Additionally, mTORC1 signaling was similar to or more pronounced in older men compared to younger men. Notably, older men displayed higher levels of amino acid transporters, nutrient sensors, and mTORC1 activators. In paper II, older men had a lesser proportion of type II fibers, smaller and misshaped type II fibers, and fewer satellite cells and capillaries surrounding their type II fibers. Additionally, older men had more denervated and “grouped” muscle fibers compared to young. In paper III, a new method (THRIFTY) for fiber typing individual fibers was developed, proving valid and more time-efficient than reference methods. In paper IV, the THRIFTY method was implemented, and the cell signaling response to intake of EAA alone and combined with REx was examined in pooled type I and type II fibers. The anabolic signaling response was similar or even more pronounced in old compared to young, with a more robust response observed in type I than in type II fibers. No deficits or alterations in autophagic signaling or E3 ligase expression were observed in older adults after EAA intake alone and combined with REx.           

In conclusion, healthy, lean, physically active, older men did not display deficits in MPS and mTORC1 signaling after anabolic cues, assessed in whole muscle and pooled type I and type II fibers. This indicates that anabolic resistance is not inherently linked to aging per se. However, older men showed increased expression of amino acid transporters, nutrient sensors, and mTORC1 activators, which may help maintain anabolic sensitivity. Despite exhibiting decrements specifically in type II fibers, such as atrophy and altered shape, there was no impairment in mTORC1 signaling or signaling related to autophagy and proteasomal degradation in these fibers after anabolic stimulation. Other factors, such as denervation and satellite cell deficits, may contribute to muscle loss in this population, but their relative impact remains unclear.

Det övergripande syftet med avhandlingen var att öka förståelsen för de cellulära och molekylära mekanismer som ligger bakom muskelförlust vid åldrande, med särskilt fokus på anabol resistens. Detta tillstånd kännetecknas av en nedsatt förmåga att stimulera proteinsyntesen vid anabola stimuli, såsom intag av proteinrik mat eller fysisk träning. En viktig bakomliggande faktor för anabol resistens är minskad aktivering av mTOR, en signalväg essentiell för cellens tillväxt. Vidare undersöktes hur åldrande påverkar egenskaperna hos snabba (typ II) och långsamma (typ I) muskelfibrer. I avhandlingen utvecklades också en ny metod för att förenkla framtida studier på enskilda muskelfibrer.           

Studierna omfattade friska, fysiskt aktiva, män i åldrarna 18–35 år och 65–74 år som genomförde ett styrketräningspass samt intog essentiella aminosyror (EAA). Muskelprover analyserades för proteinsyntes, mTOR-signalering och markörer för proteinnedbrytning. Ytterligare analyser i typ I och typ II muskelfibrer utfördes för att studera morfologi, stamceller, kapillärer och tecken på denervering för att öka förståelsen av åldrandets påverkan på dessa olika fibertyper.           

Resultatet från studie I visade att proteinsyntesen ökade efter intag av EAA, en effekt som förstärktes efter styrketräning, men inga skillnader fanns mellan unga och äldre män. Den äldre gruppen hade däremot högre mTOR-signalering och ökade nivåer av proteiner relaterade till aminosyraupptag och aktivering av mTOR-signalvägen. Studie II visade att äldre män hade en högre andel typ I fibrer, mindre och missformade typ II fibrer, samt färre stamceller och kapillärer kring typ II fibrerna. Den äldre gruppen hade också fler denerverade fibrer och en högre andel grupperade typ I fibrer. I studie III utvecklades en ny metod, THRIFTY, för snabb och effektiv fibertypning av enskilda muskelfibrer. Metoden möjliggjorde, bland annat, tillförlitlig identifiering av hybridfibrer. Studie IV visade att muskelfibrer från äldre män inte hade nedsatt mTOR-signalering eller förändringar i signalvägar relaterade till proteinnedbrytning i respons till anabola stimuli.           

Sammanfattningsvis, proteinsyntes och mTOR-signalering efter EAA-intag, med eller utan styrketräning, inte är nedsatt hos friska, fysiskt aktiva äldre män. Detta tyder på att åldrande i sig inte är en huvudorsak till anabol resistens. Friska äldre har ett högre innehåll av proteiner som aktiverar mTOR-signalvägen, vilket kan hjälpa till att bevara anabol känslighet. Muskelvävnaden från friska äldre uppvisar dock förändringar som fiberatrofi och förlust av stamceller, främst i typ II fibrer, samt tecken på denervering. Muskelatrofin som specifikt drabbar typ II fibrer vid åldrande verkar inte bero på akuta förändringar i signalvägar för proteinsyntes eller proteinnedbrytning vid anabolt stimuli, men kan potentiellt förklaras av ökad denervering eller förlust av stamceller. 

Human skeletal muscle fiber type composition varies greatly along the muscle, so one biopsy may not accurately represent the whole muscle. Recommendations on the number of biopsies and fiber counts using immunohistochemistry and whether these findings can be extrapolated to other muscles are lacking. We assessed fiber type composition in the vastus lateralis and gastrocnemius medialis muscles of 40 individuals. Per muscle, we took four biopsy samples from one incision, collecting two samples each from a proximally and distally directed needle. Based on another dataset involving 10 vastus lateralis biopsies per participant (N=7), we calculated 95% limits of agreement for subsets of biopsies and fiber counts compared to the 10-biopsy average. Average absolute differences in type I fiber proportions between proximal and distal, and between within-needle samples were 6.9 and 4.5 percentage points in the vastus lateralis, and 5.5 and 4.4 percentage points in the gastrocnemius medialis, respectively. The 95% limits of agreement narrowed to ±10 percentage points when 200 fibers from at least three biopsies were analyzed, with minimal improvements with greater fiber counts. Type I fiber proportions in the vastus lateralis and gastrocnemius medialis showed a moderate positive association (r²=0.22; p=0.006; at least 200 fibers in each of three to four samples per muscle). In conclusion, three biopsies with a minimum of 200 counted fibers are required to estimate vastus lateralis fiber type composition within ±10 percentage points. Even when using these standards, researchers should be cautious when extrapolating muscle fiber type proportions from one muscle to another.

Insulin resistance (IR) is a risk factor for the development of several major metabolic diseases. Muscle fiber composition is established early in life and is associated with insulin sensitivity. Hence, muscle fiber composition was used to identify early defects in the development of IR in healthy young individuals in the absence of clinical manifestations. Biopsies were obtained from the thigh muscle, followed by an intravenous glucose tolerance test. Indices of insulin action were calculated and cardiovascular measurements, analyses of blood and muscle were performed. Whole-body insulin sensitivity (SI_galvin) was positively related to expression of type I muscle fibers (r=0.49; P<0.001) and negatively related to resting heart rate (HR, r=-0.39; P<0.001), which was also negatively related to expression of type I muscle fibers (r=-0.41; P<0.001). Muscle protein expression of endothelial nitric oxide synthase (eNOS), whose activation results in vasodilation, was measured in two subsets of subjects expressing a high percentage of type I fibers (59±6%; HR = 57±9 beats/min; SI_galvin = 1.8±0.7 units) or low percentage of type I fibers (30±6%; HR = 71±11; SI_galvin = 0.8±0.3 units; P<0.001 for all variables vs. first group). eNOS expression was: 1. higher in subjects with high type I expression; 2. almost two-fold higher in pools of type I vs. II fibers; 3. only detected in capillaries surrounding muscle fibers; and 4. linearly associated with SI_galvin. These data demonstrate that an altered function of the autonomic nervous system and a compromised capacity for vasodilation in the microvasculature occur early in the development of IR.

The objective of this work was to investigate myonuclear permanence and transcriptional regulation as mechanisms for cellular muscle memory after strength training in humans. Twelve untrained men and women performed 10 weeks of unilateral elbow-flexor strength training followed by 16 weeks of de-training. Thereafter, 10 weeks' re-training was conducted with both arms: the previously trained arm and the contralateral untrained control arm. Muscle biopsies were taken from the trained arm before and after both training periods and from the control arm before and after re-training. Muscle biopsies were analysed for fibre cross-sectional area (fCSA), myonuclei and global transcriptomics (RNA sequencing). During the first training period, myonuclei increased in type 1 (13 ± 17%) and type 2 (33 ± 23%) fibres together with a 30 ± 43% non-significant increase in mixed fibre fCSA (P = 0.069). Following de-training, fCSA decreased in both fibre types, whereas myonuclei were maintained, resulting in 33% higher myonuclear number in previously trained vs. control muscle in type 2 fibres. Furthermore, in the previously trained muscle, three differentially expressed genes (DEGs; EGR1, MYL5 and COL1A1) were observed. Following re-training, the previously trained muscle showed larger type 2 fCSA compared to the control (P = 0.035). However, delta change in type 2 fCSA was not different between muscles. Gene expression was more dramatically changed in the control arm (1338 DEGs) than in the previously trained arm (822 DEGs). The sustained higher number of myonuclei in the previously trained muscle confirms myonuclear accretion and permanence in humans. Nevertheless, because of the unclear effect on the subsequent hypertrophy with re-training, the physiological benefit remains to be determined. KEY POINTS: Muscle memory is a cellular mechanism that describes the capacity of skeletal muscle fibres to respond differently to training stimuli if the stimuli have been previously encountered. This study overcomes past methodological limitations related to the choice of muscles and analytical procedures. We show that myonuclear number is increased after strength training and maintained during de-training. Increased myonuclear number and differentially expressed genes related to muscle performance and development in the previously trained muscle did not translate into a clearly superior responses during re-training. Because of the unclear effect on the subsequent hypertrophy and muscle strength gain with re-training, the physiological benefit remains to be determined.

Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg^-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P < 0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P < 0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P < 0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.

Cumulative evidence supports the hypothesis that hypoxia acts as a regulator of muscle mass. However, the underlying molecular mechanisms remain incompletely understood, particularly in human muscle. Here we examined the effect of hypoxia on signaling pathways related to ribosome biogenesis and myogenic activity following an acute bout of resistance exercise. We also investigated whether hypoxia influenced the satellite cell response to resistance exercise. Employing a randomized, crossover design, eight men performed resistance exercise in normoxia (FiO₂ 21%) or normobaric hypoxia (FiO₂ 12%). Muscle biopsies were collected in a time-course manner (before, 0, 90, 180 min and 24 h after exercise) and were analyzed with respect to cell signaling, gene expression and satellite cell content using immunoblotting, RT-qPCR and immunofluorescence, respectively. In normoxia, resistance exercise increased the phosphorylation of RPS6, TIF-1A and UBF above resting levels. Hypoxia reduced the phosphorylation of these targets by ~37%, ~43% and ~ 67% throughout the recovery period, respectively (p < .05 vs. normoxia). Resistance exercise also increased 45 S pre-rRNA expression and mRNA expression of c-Myc, Pol I and TAF-1A above resting levels, but no differences were observed between conditions. Similarly, resistance exercise increased mRNA expression of myogenic regulatory factors throughout the recovery period and Pax7⁺ cells were elevated 24 h following exercise in mixed and type II muscle fibers, with no differences observed between normoxia and hypoxia. In conclusion, acute hypoxia attenuates ribosome signaling, but does not impact satellite cell pool expansion and myogenic gene expression following a bout of resistance exercise in human skeletal muscle.