No abstract

ular basis of the myogenic profile of aged human skeletal muscle satellite cells during differentiation. Experimental Gerontology, Elsevier, 2009, 44 (8) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT AbstractSarcopenia is the age-related loss of muscle mass, strength and function. Human muscle proteins are synthesized at a slower rate in the elderly than in young adults, leading to atrophy and muscle mass loss with a decline in the functional capability. Additionally, aging is accompanied by a decrease in the ability of muscle tissue to regenerate following injury or overuse due to the impairment of intervening satellite cells, in which we previously reported oxidative damage evidences. The aim of the present study was to determine the effects of aging on myoblasts and myotubes obtained from human skeletal muscle, and characterize the transcriptional profile as molecular expression patterns in relation to age-dependent modifications in their regenerative capacity. Our data show that the failure to differentiate does not depend on reduced myogenic cell number, but difficulty to complete the differentiation program. Data reported here suggested the following findings: i) oxidative damage accumulation in molecular substrates, probably due to impaired antioxidant activity and insufficient repair capability, ii) limited capability of elderly myoblasts to execute a complete differentiation program; restricted fusion, possibly due to altered cytoskeleton turnover and extracellular matrix degradation, and iii) activation of atrophy mechanism by activation of a specific FOXO-dependent program.

Before establishing terminal synapses with their final muscle targets,migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twistermutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor α-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.

During aging, skeletal muscles show reduced mass and functional capacity largely due to loss of the regenerative ability of satellite cells (SCs), the quiescent stem cells located beneath the basal lamina surrounding each myofiber. While both the external environment and intrinsic properties of SCs appear to contribute to the age-related SC deficiency, the latter ones have been poorly investigated especially in humans. In the present work, we analyzed several parameters of SCs derived from biopsies of vastus lateralis muscle from healthy non-trained young (28.7±5.9 years; n=10) and aged (77.3±6.4 years; n= 11) people. Compared with young SCs, aged SCs showed impaired differentiation when cultured in differentiation medium, and exhibited the following: (1) reduced proliferation; (2) higher expression levels of S100B, a negative regulator of myoblast differentiation; (3) undetectable levels in growth medium of fulllength RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily, the engagement of which enhances myoblast differentiation; and (4) lower expression levels of the transcription factors, MyoD and Pax7. Also, either overexpression of full-length RAGE or knockdown of S100B in aged SCs resulted in enhanced differentiation, while overexpression of either a nontransducing mutant of RAGE (RAGEΔcyto) or S100B in young SCs resulted in reduced differentiation compared with controls. Moreover, while aged SCs maintained the ability to respond to mitogenic factors (e.g., bFGF and S100B), they were no longer able to secrete these factors, unlike young SCs. These data support a role for intrinsic factors, besides the extracellular environment in the defective SC function in aged skeletal muscles.

In humans aging is a complex process that determines many physical and metabolic alterations correlated to the accumulation of oxidative damage in different tissues. Sarcopenia is an age-related nonpathological condition that includes a progressive loss of mass and strength in skeletal muscle, associated with a decline in the fibers' functional capability. This condition could be correlated to abnormal reactive oxygen species (ROS) accumulation with consequent fiber oxidative damage. This complex situation is not only evident in mature muscle fibers but also in muscle resident satellite cells (involved in fiber damage repairing) in which some functional parameters, at least for that concerns the Ca(2+) homeostasis, seem to be modified. In fact, our data show that there is an age-dependent increase of lipid peroxidation, in cultured myotubes (differentiated and fused satellite cells) after 7 days of in vitro differentiation. In these substrates also the capacity of these cells to produce Ca(2+) transient in response to various stimuli (ATP, caffeine, nicotine, KCl) is, sometimes, drastically modified. In particular, the presence of an age-dependent defective status of excitation-contraction (EC) coupling apparatus is supported by a single cell Ca(2+) analysis obtained from myotubes (derived from aged muscles) in the presence of 40 mM caffeine or 40 mM KCl. The alkaloid presence induces a complete emptying of ryanodine-dependent calcium stores indicating a probable integrity both of SR-terminal cisternae and/or the specific Ca(2+) channel known as RyR1. However, if a sarcolemmal depolarization is induced by the addition of 40 mM KCl in the experimental medium then Ca(2+) release RyR1-dependent can be observed only if Ca(2+) is present in the experimental solution. These results suggest that the EC uncoupling status could be due to the alteration of the interaction between RyR and DHPR. The two receptors are present and functionally active in myotubes from aged donors but they are probably still not in the right localization. These results suggest that during donor's life the satellite cells undergo an aging process similar to the one observed in skeletal muscle tissue, even if they are in a quiescence status for most of the time.