Abigail L. Mackey

Molecular aging and rejuvenation of human muscle stem cells

Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short‐lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans. Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth factor beta (TGF‐β)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen‐activated protein kinase (MAPK)/phosphate extracellular signal‐regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular understanding, combined with data that human satellite cells remain intrinsically young, introduced novel therapeutic targets. Indeed, activation of MAPK/Notch restored ‘youthful’ myogenic responses to satellite cells from 70‐year‐old humans, rendering them similar to cells from 20‐year‐old humans. These findings strongly suggest that aging of human muscle maintenance and repair can be reversed by ‘youthful’ calibration of specific molecular pathways.

Differentially Activated Macrophages Orchestrate Myogenic Precursor Cell Fate During Human Skeletal Muscle Regeneration

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti‐inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin‐positive MPCs were preferentially coupled to MPs harboring anti‐inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair. STEM CELLS2013;31:384–396

Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis

In skeletal muscle and tendon the extracellular matrix confers important tensile properties and is crucially important for tissue regeneration after injury. Musculoskeletal tissue adaptation is influenced by mechanical loading, which modulates the availability of growth factors, including growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I), which may be of key importance. To test the hypothesis that GH promotes matrix collagen synthesis in musculotendinous tissue, we investigated the effects of 14 day administration of 33–50 μg kg−1 day−1 recombinant human GH (rhGH) in healthy young individuals. rhGH administration caused an increase in serum GH, serum IGF‐I, and IGF‐I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9‐fold and 1.3‐fold, respectively (P < 0.01 and P= 0.02), and muscle collagen I mRNA expression and muscle collagen protein synthesis increased by 2.3‐fold and 5.8‐fold, respectively (P < 0.01 and P= 0.06). Myofibrillar protein synthesis was unaffected by elevation of GH and IGF‐I. Moderate exercise did not enhance the effects of GH manipulation. Thus, increased GH availability stimulates matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue.

Local NSAID infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise

Despite the widespread consumption of nonsteroidal anti-inflammatory drugs (NSAIDs), the influence of these drugs on muscle satellite cells is not fully understood. The aim of the present study was to investigate the effect of a local NSAID infusion on satellite cells after unaccustomed eccentric exercise in vivo in human skeletal muscle. Eight young healthy males performed 200 maximal eccentric contractions with each leg. An NSAID was infused via a microdialysis catheter into the vastus lateralis muscle of one leg (NSAID leg) before, during, and for 4.5 h after exercise, with the other leg working as a control (unblocked leg). Muscle biopsies were collected before and 8 days after exercise. Changes in satellite cells and inflammatory cell numbers were investigated by immunohistochemistry. Satellite cells were identified using antibodies against neural cell adhesion molecule and Pax7. The number of Pax7(+) cells per myofiber was increased by 96% on day 8 after exercise in the unblocked leg (0.14 +/- 0.04, mean +/- SE) compared with the prevalue (0.07 +/- 0.02, P < 0.05), whereas the number of Pax7(+) cells was unchanged in the leg muscles exposed to the NSAID (0.07 +/- 0.01). The number of inflammatory cells (CD68(+) or CD16(+) cells) was not significantly increased in either of the legs 8 days after exercise and was unaffected by the NSAID. The main finding in the present study was that the NSAID infusion for 7.5 h during the exercise day suppressed the exercise-induced increase in the number of satellite cells 8 days after exercise. These results suggest that NSAIDs negatively affect satellite cell activity after unaccustomed eccentric exercise.

Enhanced satellite cell proliferation with resistance training in elderly men and women.

In addition to the well‐documented loss of muscle mass and strength associated with aging, there is evidence for the attenuating effects of aging on the number of satellite cells in human skeletal muscle. The aim of this study was to investigate the response of satellite cells in elderly men and women to 12 weeks of resistance training. Biopsies were collected from the m. vastus lateralis of 13 healthy elderly men and 16 healthy elderly women (mean age 76±SD 3 years) before and after the training period. Satellite cells were visualized by immunohistochemical staining of muscle cross‐sections with a monoclonal antibody against neural cell adhesion molecule (NCAM) and counterstaining with Mayers hematoxylin. Compared with the pre‐training values, there was a significant increase (P<0.05) in the number of NCAM‐positively stained cells per fiber post‐training in males (from 0.11±0.03 to 0.15±0.06; mean±SD) and females (from 0.11±0.04 to 0.13±0.05). These results suggest that 12 weeks of resistance training is effective in enhancing the satellite cell pool in skeletal muscle in the elderly.

Assessment of satellite cell number and activity status in human skeletal muscle biopsies

The primary aim of our study was to validate the assessment of myonuclear and satellite cell number in biopsies from human skeletal muscle. We found that 25 type I and 25 type II fibers are sufficient to estimate the mean number of myonuclei per fiber. In contrast, the assessment of satellite cells improved when more fibers were included. Second, we report that small differences in counting satellite cells using CD56 and Pax7 antibodies can be attributed to the different staining profiles. Third, we provide support for the use of Ki67 in evaluating the proportion of active satellite cells. We observed very few (up to 1.3%) active satellite cells in healthy adult skeletal muscle at rest, but they increased significantly (up to 7‐fold) following muscle activity. This study provides valuable tools to assess the behavior of satellite cells, both in pathological conditions and in response to physiological stimuli. Muscle Nerve 40: 455–465, 2009

Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle

The purpose of this study was to test the hypothesis that remodeling of skeletal muscle extracellular matrix (ECM) is involved in protecting human muscle against injury. Biopsies were obtained from medial gastrocnemius muscles after a single bout of electrical stimulation (B) or a repeated bout (RB) 30 d later, or 30 d after a single stimulation bout (RBc). A muscle biopsy was collected from the control leg for comparison with the stimulated leg. Satellite cell content, tenascin C, and muscle regeneration were assessed by immunohistochemistry; real‐time PCR was used to measure mRNA levels of collagens, laminins, heat‐shock proteins (HSPs), inflammation, and related growth factors. The large responses of HSPs, CCL2, and tenascin C detected 48 h after a single bout were attenuated in the RB trial, indicative of protection against injury. Satellite cell content and 12 target genes, including IGF‐1, were elevated 30 d after a single bout. Among those displaying the greatest difference vs. control muscle, ECM laminin‐β1 and collagen types I and III were elevated ~6‐ to 9‐fold (P<0.001). The findings indicate that the sequenced events of load‐induced early deadhesion and later strengthening of skeletal muscle ECM play a role in protecting human muscle against future injury.—Mackey, A. L., Brandstetter, S., Schjerling, P., Bojsen‐Moller, J., Qvortrup, K., Pedersen, M. M., Doessing, S. Kjaer, M., Magnus‐son, S. P., Langberg, H. Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle. FASEB J. 25, 1943‐1959 (2011). www.fasebj.org

Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging. Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross‐links and a buildup of advanced glycation end‐product cross‐links. Altered mechanotransduction, poorer activation of satellite cells, poorer chemotactic and delayed inflammatory responses, and a change in modulators of the ECM are important cellular changes. It is possible that the structural and biochemical changes in skeletal muscle ECM contribute to the increased stiffness and impairment in force generated by the contracting muscle fibers seen with aging. The cellular interactions provide and potentially coordinate an adaptation to mechanical loading and ensure successful regeneration after muscle injury. Some of the changes in skeletal muscle ECM with aging may be preventable with resistance or weight training, but it is clear that more human studies are needed on the topic.

Protein-containing nutrient supplementation following strength training enhances the effect on muscle mass, strength, and bone formation in postmenopausal women

We evaluated the response of various muscle and bone adaptation parameters with 24 wk of strength training in healthy, early postmenopausal women when a nutrient supplement (protein, carbohydrate, calcium, and vitamin D) or a placebo supplement (a minimum of energy) was ingested immediately following each training session. At inclusion, each woman was randomly and double-blindedly assigned to a nutrient group or a placebo (control) group. Muscle hypertrophy was evaluated from biopsies, MRI, and dual-energy X-ray absorptiometry (DEXA) scans, and muscle strength was determined in a dynamometer. Bone mineral density (BMD) was measured using DEXA scans, and bone turnover was determined from serum osteocalcin and collagen type I cross-linked carboxyl terminal peptide. The nutrient group improved concentric and isokinetic (60 degrees /s) muscle strength from 6 to 24 wk by 9 +/- 3% (P < 0.01), whereas controls showed no change (1 +/- 2%, P > 0.05). Only the nutrient group improved lean body mass (P < 0.05) over the 24 wk. BMD responded similarly at the lumbar spine but changed differently in the two groups at the femoral neck (P < 0.05) [control: 0.943 +/- 0.028 to 0.930 +/- 0.024 g/mm(3) (-1.0 +/- 1.4%); nutrient group: 0.953 +/- 0.051 to 0.978 +/- 0.043 g/mm(3) (3.8 +/- 3.4%)] when adjusted for age, body mass index, and BMD at inclusion. Bone formation displayed an interaction (P < 0.05), mainly caused by increased osteocalcin at 24 wk in the nutrient group. In conclusion, we report that nutrient supplementation results in superior improvements in muscle mass, muscle strength, femoral neck BMD, and bone formation during 24 wk of strength training. The observed differences following such a short intervention emphasize the significance of postexercise nutrient supply on musculoskeletal maintenance.

Evidence of skeletal muscle damage following electrically stimulated isometric muscle contractions in humans.

It is unknown whether muscle damage at the level of the sarcomere can be induced without lengthening contractions. To investigate this, we designed a study where seven young, healthy men underwent 30 min of repeated electrical stimulated contraction of m. gastrocnemius medialis, with the ankle and leg locked in a fixed position. Two muscle biopsies were collected 48 h later: one from the stimulated muscle and one from the contralateral leg as a control. The biopsies were analyzed immunohistochemically for inflammatory cell infiltration and intermediate filament disruption. Ultrastructural changes at the level of the z-lines were investigated by transmission electron microscopy. Blood samples were collected for measurement of creatine kinase activity, and muscle soreness was assessed in the days following stimulation. The biopsies from the stimulated muscle revealed macrophage infiltration and desmin-negative staining in a small percentage of myofibers in five and four individuals, respectively. z-Line disruption was evident at varying magnitudes in all subjects and displayed a trend toward a positive correlation (r = 0.73, P = 0.0663) with the force produced by stimulation. Increased muscle soreness in all subjects, combined with a significant increase in creatine kinase activity (P < 0.05), is indirectly suggestive of muscle damage, and the novel findings of the present study, i.e., 1) macrophages infiltration, 2) lack of desmin staining, and 3) z-line disruption, provide direct evidence of damage at the myofiber and sarcomere levels. These data support the hypothesis that muscle damage at the level of the sarcomere can be induced without lengthening muscle contractions.