Key points• Elderly individuals require a prolonged recovery phase in order to return to initial muscle mass levels following short-term immobilisation.• The cellular mechanisms responsible for the attenuated re-growth and associated molecular signalling processes in ageing human skeletal muscle are not fully understood.• The main study finding was the observation of a less marked muscle mass recovery after immobilisation in elderly compared to young individuals that was paralleled by an elevation in myogenic precursor cell content in young individuals only, whereas the elderly failed to demonstrate any change in myogenic precursor cells.• No age-related differences were observed in the expression of major myogenic regulating factors known to promote skeletal muscle hypertrophy or satellite cell proliferation (IGF-1Ea, MGF, MyoD1, myogenin, HGF gene products).• In contrast, the expression of myostatin demonstrated a more pronounced up-regulation following immobilisation along with an attenuated down-regulation in response to reloading in older compared to young individuals, which may have contributed to the present lack of satellite cell proliferation in ageing muscle.Abstract Recovery of skeletal muscle mass from immobilisation-induced atrophy is faster in young than older individuals, yet the cellular mechanisms remain unknown. We examined the cellular and molecular regulation of muscle recovery in young and older human subjects subsequent to 2 weeks of immobility-induced muscle atrophy. Retraining consisted of 4 weeks of supervised resistive exercise in 9 older (OM: mean age) 67.3, range 61-74 yrs) and 11 young (YM: mean age 24.4, range 21-30 yrs) males. Measures of myofibre area (MFA), Pax7-positive satellite cells (SCs) associated with type I and type II muscle fibres, as well as gene expression analysis of key growth and transcription factors associated with local skeletal muscle milieu, were performed after 2 weeks immobility (Imm) and following 3 days (+3d) and 4 weeks (+4wks) of retraining. OM demonstrated no detectable gains in MFA (vastus lateralis muscle) and no increases in number of Pax7-positive SCs following 4wks retraining, whereas YM increased their MFA (P < 0.05), number of Pax7-positive cells, and had more Pax7-positive cells per type II fibre than OM at +3d and +4wks (P < 0.05). No age-related differences were observed in mRNA expression of IGF-1Ea, MGF, MyoD1 and HGF with retraining, whereas myostatin expression levels were more down-regulated in YM compared to OM at +3d (P < 0.05). In conclusion, the diminished muscle re-growth after immobilisation in elderly humans was associated with a lesser response in satellite cell proliferation in combination with an age-specific regulation of myostatin. In contrast, expression of local growth factors did not seem to explain the age-related difference in muscle mass recovery.

Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2–4 days) of human disuse-muscle atrophy along with a marked reduction in PGC-1α and PGC-1β (1–4 days) and a ∼10% decrease in myofiber size (4 days). Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1–4 days) of immobilization. In contrast, Akt phosphorylation was unchanged in old muscle after 2 days and increased after 4 days of immobilization. Further, an age-specific down-regulation of MuRF-1 and Atrogin-1 expression levels was observed following 2 weeks of immobilization, along with a slowing atrophy response in aged skeletal muscle. Neither the immediate loss of muscle mass, nor the subsequent age-differentiated signaling responses could be explained by changes in inflammatory mediators, apoptosis markers or autophagy indicators. Collectively, these findings indicate that the time-course and regulation of human skeletal muscle atrophy is age dependent, leading to an attenuated loss in aging skeletal muscle when exposed to longer periods of immobility-induced disuse.

Delta like 1 (DLK1) has been proposed to act as a regulator of cell fate determination and is linked to the development of various tissues including skeletal muscle. Herein we further investigated DLK1 expression during skeletal muscle remodeling. Although practically absent in normal adult muscle, DLK1 was upregulated in all human myopathies analyzed, including Duchenne-and Becker muscular dystrophies. Substantial numbers of DLK1 1 satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1 1 cells were identified during muscle regeneration in animal models in which the peak expression of Dlk1 mRNA and protein coincided with that of myoblast differentiation and fusion. In addition to perivascular DLK11 cells, interstitial DLK1 1 cells were numerous in regenerating muscle, and in agreement with colocalization studies of DLK1 and CD90/DDR2, qPCR of fluorescence-activated cell sorting DLK1 1 and DLK1 2 cells revealed that the majority of DLK11 cells isolated at day 7 of regeneration had a fibroblast-like phenotype. The existence of different DLK11 populations was confirmed in cultures of primary derived myogenic cells, in which large flat nonmyogenic DLK11 cells and small spindle-shaped cells coexpressing DLK1 and muscle-specific markers were observed. Myogenic differentiation was achieved when sorted DLK11 cells were cocultured together with primary myoblasts revealing a myogenic potential that was 10% of the DLK1 2 population. Transplantation of DLK1 1 cells into lacerated muscle did, however, not give rise to DLK1 1 cell-derived myofibers. We suggest that the DLK1 1 subpopulations identified herein each may contribute at different levels/time points to the processes involved in muscle development and remodeling. STEM CELLS 2009;27:898-908 Disclosure of potential conflicts of interest is found at the end of this article.

S U M M A R Y Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15-16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment.