Orietta Pansarasa

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle

This study was conducted in order to provide evidence for the role of reactive oxygen species (ROS) in human skeletal muscle aging. We used human muscle samples obtained from hospitalized patients in an open study with matched pairs of individuals of different ages. The subjects, ranging in age from 17 to 91 years, were grouped as follows: 17-25-, 26-35-, 36-45-, 46-55-, 56-65-, 66-75-, 76-85-, and 86-91-year-old groups. To investigate the relationship between muscle aging and oxidative damage we measured total and Mn-dependent superoxide dismutase (total SOD, MnSOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; total reduced and oxidized glutathione (GSHtot, GSH, and GSSG) levels; lipid peroxidation (LPO), and protein carbonyl content (PrC). Total SOD activity decreases significantly with age in the 66-75-year-old group, although MnSOD activity increases significantly in the 76-85-year-old group. The activity of the two H2O2 detoxifying enzymes (GSHPx and CAT) did not change with age, as do GSHtot and GSH levels. GSSG levels increased significantly (76-85- and 86-91-year-old groups) with age. We observed a significant increase in LPO levels (66-75- and 76-85-year-old groups), although the PrC content shows a trend of increase without gaining the statistical significance. These results support the idea that ROS play an important role in the human muscle aging process.

Autologous transplantation of muscle-derived CD133(+) stem cells in Duchenne muscle patients

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Age and sex differences in human skeletal muscle: role of reactive oxygen species.

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17–40 years), adult (41–65 years) and aged (66–91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66–91 year-old vs. the 17–40 year-old men; MnSOD activity was significantly greater in 66–91 year-old vs. 17–40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41–65 and 66–91 year-old vs. the 17–40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.

In vitro antioxidant properties of amifostine (WR-2721, ethyol)

Purpose: Amifostine (WR-2721), a phosphorylated aminothiol pro-drug which is an analogue of cysteamine, is a selective cytoprotective agent for normal tissues from the toxicities associated with chemotherapy and irradiation. Despite a growing number of reports strongly supporting amifostines clinical efficacy, few authors have focused on the biochemical basis of amifostines antioxidant activity. Methods: We report on amifostines free-radical scavenging activity against superoxide (O2˙−), hydroxyl (OH−) and lipoperoxyl radicals in an in vitro model, using pure chemical systems. Amifostine was dephosphorylated to its active metabolite, WR-1065, by adding 10% non-heat-inactivated serum; different amifostine concentrations (1, 10, 50, 100 μM and 200 μM) and pH conditions (pH 5, 7.4 and 9) were tested. Results: Independent of the concentration, amifostine exhibited no major activity against O2˙− ions, neither did any pH variations in the experimental model provide any scavenger effects of the drug against O2˙− radicals. On the other hand, the protective effect of amifostine against OH− radicals was confirmed, yielding an EC50 of 255 μM at pH 7.4 and 230 μM at pH 5. Finally, amifostine exhibited scavenging activity against spontaneous lipoperoxidation, but no apparent antioxidant effect on iron ascorbate-induced lipoperoxidation. Conclusions: With this in vitro study, we are able to confirm the scavenging activity of the chemo- and radioprotector amifostine, whose activity seems to be particularly important from a biological point of view, since it is exerted mainly against highly reactive OH−.

Oxidative damage after severe head injury and its relationship to neurological outcome

OBJECTIVE We sought to establish the time course of reactive oxygen species after severe head injuries in humans and to investigate their relationship with clinical outcomes. METHODS Both the markers of oxidative damage—malonylaldehyde (MDA) and the enzymatic and nonenzymatic antioxidant defenses (i.e., superoxide dismutase [SOD] and vitamin E [VE], respectively)—were studied. To assess the time course of MDA, SOD, and VE, jugular bulb (JB) and peripheral venous blood samples were obtained from 30 patients within 8 hours of severe head trauma onset (T0) and 6 (T1), 12 (T2), 24 (T3), and 48 hours (T4) after trauma onset. Patients were divided into good and poor outcome groups according to their 6-month neurological outcome as determined on the basis of their Glasgow Outcome Scale scores and biochemical profiles. RESULTS In JB samples, MDA levels increased significantly at T1, T2, T3, and T4 as compared with T0; SOD activity increased significantly at T2 and T3 as compared with T0; and VE levels decreased significantly at T1, T2, and T3 as compared with T0. The same variables did not change significantly over time in peripheral venous blood samples. Moreover, the MDA levels and SOD activity detected in JB samples were significantly higher in the poor outcome group at T1 and T2. No significant difference in VE levels was observed between the two outcome groups. CONCLUSION Reactive oxygen species-mediated oxidative damage can play an important role in determining the prognosis of severe brain injury in humans.

Oral Amino Acid Supplementation Counteracts Age-Induced Sarcopenia in Elderly Rats

We investigated the effects of a specific mixture of amino acid (AA) supplements on the adaptation changes induced by aging in the soleus muscle of rats. Male Wistar rats were divided into 3 groups (n = 5 each): young control (YO), 3 months of age; elderly control (EL), 18 months of age; and elderly orally supplemented with an AA mixture (EL-AA), 18 months of age, given as 0.1 g/kg per day in drinking water for 8 weeks. Myosin heavy chain (MHC) composition was analyzed in all muscles. The total fiber number and fiber cross-sectional area of types 1 and 2A fibers were also measured in immunostained sections of the soleus muscle. The ratios between the sarcomere volume (Vsar) and the total volume (Vtot) and single muscle fibers were studied by electron microscopy. The expression of total and phosphorylated serine/threonine protein kinase mammalian target of rapamycin (mTOR), a potent regulator of messenger RNA translation initiation, was also determined in all groups. Aging was associated with an overall shift toward the expression of a slower MHC phenotype, atrophy of fast and slow fibers, a significant decrease in Vtot/Vsar, and no changes in total fiber number. AA supplementation antagonized the effects of aging. A shift toward the expression of faster MHC isoforms was observed. Fiber atrophy appeared to be partly counteracted by the AA supplements; we noted an increase in cross-sectional area fibers and Vtot/Vsar in EL-AAs. Total and phosphorylated mTOR expression appeared to decrease in EL and was restored by the AA supplements. Collectively, these results suggest that aging-induced muscle adaptations can be partly restored by AA supplementation. An mTOR signal pathway may mediate the effects on fiber trophism.

Corrigendum: Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs

This corrects the article DOI: 10.1038/nature05282

Resistance training of long duration modulates force and unloaded shortening velocity of single muscle fibres of young women

The aim of the present study was to clarify the impact of long term (1 year) resistance training (RT) on structure and function of single muscle fibres of vastus lateralis in young female subjects. Five young women (age: 25.4+/-6.2 year) performed exercise sessions at 60% of single subject own repetition maximum (1 RM) 1h twice a week. Maximum voluntary force was determined pre- and post-RT and was found to significantly increase post-RT ensuring a successful impact of RT on muscle performance in vivo. Needle muscle biopsy samples were obtained both pre- and post-RT and the following determinations were performed: myosin heavy chain isoform (MHC) distribution of the whole muscle samples by SDS-PAGE; cross sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n=358) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE. The results suggest that the long duration of RT can determine a significant increase in specific force (Po/CSA) and unloaded shortening velocity (Vo) of single muscle fibres in female subjects, whereas no muscle fibre hypertrophy and no shift in MHC isoform content was observed.

Structural and functional alterations of muscle fibres in the novel mouse model of facioscapulohumeral muscular dystrophy

We recently generated a mouse model of facioscapulohumeral muscular dystrophy (FSHD) by selectively overexpressing FRG1, a candidate gene for FSHD, in skeletal muscle. The muscles of the FRG‐1 mice did not show any plasmamembrane defect suggesting a novel pathogenetic mechanism for FSHD. Here, we study structure and function of muscle fibres from three lines of mice overexpressing FRG1 at different levels: FRG1‐low, FRG1‐med, FRG1‐high. Cross‐sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of identified types of muscle fibres from FRG1‐low and FRG1‐med mice were analysed and found to be lower than in WT mice. Fast fibres and especially type 2B fibres (the fastest type) were preferentially involved in the dystrophic process showing a much larger force deficit than type 1 (slow) fibres. Consistent with the latter observation, the MHC isoform distribution of several muscles of the three FRG1 lines showed a shift towards slower MHC isoforms in comparison to WT muscle. Moreover, fast muscles showed a more evident histological deterioration, a larger atrophy and a higher percentage of centrally nucleated fibres than the soleus, the slowest muscle in mice. Interestingly, loss in CSA, Po/CSA and Vo of single muscle fibres and MHC isoform shift towards a slower phenotype can be considered early signs of muscular dystrophy (MD). They were, in fact, found also in FRG1‐low mice which did not show any impairment of function in vivo and of muscle size in vitro and in soleus muscles, which had a completely preserved morphology. This study provides a detailed characterization of structure and function of muscle fibres in a novel murine model of one of the main human MDs and suggests that fundamental features of the dystrophic process, common to most MDs, such as the intrinsic loss of contractile strength of muscle fibres, the preferential involvement of fast fibres and the shift towards a slow muscle phenotype can occur independently from obvious alterations of the plasma membrane.