Rosa Mancinelli

Molecular basis of the myogenic profile of aged human skeletal muscle satellite cells during differentiation

Sarcopenia 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.

Specific correlations between muscle oxidative stress and chronic fatigue syndrome: a working hypothesis

Chronic fatigue syndrome (CFS) is a relatively common disorder defined as a status of severe persistent disabling fatigue and subjective unwellness. While the biological basis of the pathology of this disease has recently been confirmed, its pathophysiology remains to be elucidated. Moreover, since the causes of CFS have not been identified, treatment programs are directed at symptom relief, with the ultimate goal of the patient regaining some level of pre-existing function and well-being. Several studies have examined whether CFS is associated with: (i) a range of infectious agents and or immune disturbance; (ii) specific changes of activity in the central or peripheral nervous systems; and (iii) elevated stress periods, which may be associated with the pathology via genetic mechanisms. The role of oxidative stress in CFS is an emerging focus of research due to evidence of its association with some pathological features of this syndrome. New data collectively support the presence of specific critical points in the muscle that are affected by free radicals and in view of these considerations, the possible role of skeletal muscle oxidative imbalance in the genesis of CFS is discussed.

Stem Cell Ageing and Apoptosis

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.

Extracellular guanosine-5′-triphosphate modulates myogenesis via intermediate Ca2+-activated K+ currents in C2C12 mouse cells

In this study we investigated the role of extracellular 5′‐guanosine‐triphosphate (GTP) on early phases of skeletal muscle differentiation using the widely used C2C12 mouse cells as a myogenic model. We show that extracellular GTP binding to specific sites activates a metabotropic cascade that leads to a transient intracellular Ca2+ mobilization, consequent activation of the intermediate Ca2+‐activated K+ channels (IKCa), and hyperpolarization of the plasma membrane. We further show that in differentiating C2C12 myoblasts GTP induces a proliferative boost, and increases the number of cells positive for the myosin heavy chain (MyHC) proteins. These effects were shown to be mediated by the IKCa channel‐dependent hyperpolarization, as evidenced by their disappearance when myoblasts were incubated with the IKCa channel inhibitor charybdotoxin. These data give new insights into nucleotide purinergic signalling pathways, and address the role of the GTP‐dependent IKCa channel activation and hyperpolarization in myogenesis.

Dual role of the caspase enzymes in satellite cells from aged and young subjects

Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage.

Tiny percutaneous needle biopsy: An efficient method for studying cellular and molecular aspects of skeletal muscle in humans

Needle biopsy is widely used to obtain specimens for physiological, anatomical and biochemical studies of skeletal muscle (SM). We optimized a procedure which we termed tiny percutaneous needle biopsy (TPNB), to efficiently gather good numbers of human satellite cells and single dissociated fibers for the functional study of skeletal muscle; these samples permit isolation of high-quality RNA and sufficient amounts of proteins to allow molecular analysis. Moreover, TPNB showed a clear advantage in that the technique was easier than other procedures used on healthy volunteers in human trials. TPNB is a very safe minor surgical procedure. It is less traumatic than needle aspiration biopsy, and significant complications are improbable. TPNB should become established as an important tool in the investigation of SM and may be employed to study various physiological aspects of SM in human subjects. We suggest that TPNB should also be used in the study of muscle diseases and disorders including muscular dystrophy, congenital myopathy, and metabolic defects.

Low Intensity Exercise Training Improves Skeletal Muscle Regeneration Potential

Purpose: The aim of this study was to determine whether 12 days of low-to-moderate exercise training at low altitude (598 m a.s.l.) improves skeletal muscle regeneration in sedentary adult women. Methods: Satellite cells were obtained from the vastus lateralis skeletal muscle of seven women before and after this exercise training at low altitude. They were investigated for differentiation aspects, superoxide anion production, antioxidant enzymes, mitochondrial potential variation after a depolarizing insult, intracellular Ca2+ concentrations, and micro (mi)RNA expression (miR-1, miR-133, miR-206). Results: In these myogenic populations of adult stem cells, those obtained after exercise training, showed increased Fusion Index and intracellular Ca2+ concentrations. This exercise training also generally reduced superoxide anion production in cells (by 12–67%), although not in two women, where there was an increase of ~15% along with a reduced superoxide dismutase activity. miRNA expression showed an exercise-induced epigenetic transcription profile that was specific according to the reduced or increased superoxide anion production of the cells. Conclusions: The present study shows that low-to-moderate exercise training at low altitude improves the regenerative capacity of skeletal muscle in adult women. The differentiation of cells was favored by increased intracellular calcium concentration and increased the fusion index. This low-to-moderate training at low altitude also depicted the epigenetic signature of cells.

Myomir dysregulation and reactive oxygen species in aged human satellite cells

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

Neuromuscular electrical stimulation improves skeletal muscle regeneration through satellite cell fusion with myofibers in healthy elderly subjects

The aim of this study was to determine whether neuromuscular electrical stimulation (NMES) affects skeletal muscle regeneration through a reduction of oxidative status in satellite cells of healthy elderly subjects. Satellite cells from the vastus lateralis skeletal muscle of 12 healthy elderly subjects before and after 8 wk of NMES were allowed to proliferate to provide myogenic populations of adult stem cells [myogenic precursor cells (MPCs)]. These MPCs were then investigated in terms of their proliferation, their basal cytoplasmic free Ca2+ concentrations, and their expression of myogenic regulatory factors (PAX3, PAX7, MYF5, MYOD, and MYOG) and micro-RNAs (miR-1, miR-133a/b, and miR-206). The oxidative status of these MPCs was evaluated through superoxide anion production and superoxide dismutase and glutathione peroxidase activities. On dissected single skeletal myofibers, the nuclei were counted to determine the myonuclear density, the fiber phenotype, cross-sectional area, and tension developed. The MPCs obtained after NMES showed increased proliferation rates along with increased cytoplasmic free Ca2+ concentrations and gene expression of MYOD and MYOG on MPCs. Muscle-specific miR-1, miR-133a/b, and miR-206 were upregulated. This NMES significantly reduced superoxide anion production, along with a trend to reduction of superoxide dismutase activity. The NMES-dependent stimulation of muscle regeneration enhanced satellite cell fusion with mature skeletal fibers. NMES improved the regenerative capacity of skeletal muscle in elderly subjects. Accordingly, the skeletal muscle strength and mobility of NMES-stimulated elderly subjects significantly improved. NMES may thus be further considered for clinical or ageing populations.NEW & NOTEWORTHY The neuromuscular electrical stimulation (NMES) effect on skeletal muscle regeneration was assessed in healthy elderly subjects for the first time. NMES improved the regenerative capacity of skeletal muscle through increased myogenic precursor cell proliferation and fusion with mature myofibers. The increased cytoplasmic free Ca2+ concentration along with MYOD, MYOG, and micro-RNA upregulation could be related to reduced O2·- production, which, in turn, favors myogenic regeneration. Accordingly, the skeletal muscle strength of NMES-stimulated lower limbs of healthy elderly subjects improved along with their mobility.

Dental pulp stem cells grown on dental implant titanium surfaces: An in vitro evaluation of differentiation and microRNAs expression.

The surface roughness of dental implants influences the proliferation and differentiation rate of adult mesenchymal stem cells (MSCs). The aim of the present study was to evaluate whether specifically treated titanium implant surfaces influenced human dental pulp stem cells (DPSCs) differentiation in an osteogenic pattern through modulation of microRNAs expression. The degree of differentiation was evaluated after 7, 14, and 21 days, through the expression of microRNAs characterizing the osteogenesis (miR-133 and miR-135), of Runx2 and Smad5 (key factor transcriptions associated with osteoblast differentiation) and Osteocalcin, marker for the bone formation process. DPSCs were cultured on sandblasted and acid-etched titanium disks, with (Test) or without the presence of ions (Control). Early differentiation of DPSCs cultured on titanium could be detected at all the evaluated time points, respect to cells grown alone. Moreover, the Test surfaces seemed to induce a more marked cells differentiation. The obtained results demonstrated that microRNAs played a pivotal role in the differentiation of MSCs and could be used as marker of osteogenic differentiation. Furthermore, the evaluated ionized sandblasted and acid-etched surface seemed to markedly enhance the development of osteoblast cells. A faster osseointegration could be achieved in the presence of specifically treated implant surfaces, promising encouraging clinical outcomes.