Ugo Carraro

Apoptosis of skeletal muscles during development and disease.

Cells from multicellular organisms self-destroy when no longer needed or when damaged. They do this by activating genetically controlled machineries that lead to apoptosis. Skeletal muscles in adult animals are fully differentiated syncytial cells. Apoptosis has been described in developing and, recently, in adult skeletal muscle. The cellular and molecular aspects of myoblast and myofibre apoptosis and their role in disease are analysed in this review. Alterations in the pathways that regulate myoblasts proliferation/differentiation lead to induction of apoptosis during myogenesis both in vivo and in vitro. In adult muscle myofibres apoptosis seems to start from segmental areas of myofibres often producing loss of a single myonucleus. The bcl2/bax system is active in muscle when apoptosis occurs. On the other hand conflicting results are reported on the role played by FasL/Fas system. These findings are confirmed by in vitro results on myotubes and on their susceptibility to apoptosis. Though apoptosis has been shown to occur in the skeletal muscle, the role played in diseases and the pattern followed in myogenic cells are far from being clear.

Apoptosis, DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise

The current view indicates that after eccentric exercise myofibers are mechanically damaged and therefore an inflammatory and necrotic process occurs. In the present paper we examine the possibility that apoptosis plays a role in normal and dystrophin‐deficient muscles after running. We analysed for apoptosis normal and dystrophin‐deficient mouse muscles after a night of spontaneous wheel‐running followed by two days of rest. Terminal deoxynucleotidyl transferase‐mediated endlabeling of DNA in nuclei in tissue sections and gel electrophoresis of extracted DNA showed the presence of fragmented DNA. Furthermore, ubiquitin, a protein whose appearance is related to apoptosis, increased in muscles of both dystrophic and normal runner mice. The present findings which confirm that DNA damage is absent in muscles of sedentary mice but present in muscles of runner mice offer a new hypothesis on early events of muscle damage.

Macrophage-released Factor Stimulates Selectively Myogenic Cells in Primary Muscle Culture

Myolibers arc reconstituted by the proliferation and fusion of muscle precursor cells when skeletal muscle is injured. One of the critical events is the peak accumulation of macrophages after 48 hours at the damage site before the satellite cell proliferation. In addition to their well-known role as a scavenger cell, there is now direct evidence of a mitogenic role of macrophages in regenerating muscle. We have utilized an in vitro model to directly investigate and prove that macrophages increase myoblast growth not only of satellite cells, but also of primary myoblasts. Rat muscle cells were cultured in the presence or absence of exudate macrophages obtained by peritoneal washing after thioglycollate broth injection. Macrophage cocullure increases several times the myoblasts/myotubes yield. This effect is particularly evident in muscle culture conditions in which fibroblast growth is predominant over myoblast proliferation, suggesting a myoblast selective mitogenic effect of macrophages. The results are confirmed by quantitative analyses of both DNA and skeletal muscle-specific-contractile proteins by gel electrophoresis and immunocytochemistry. Experiments with macrophage-conditioned media show this effect is mediated by soluble factors. This growth factor-like activity, which has been shown to be acid-stable and heatlabile, exerts its effects not only on specialized satellite cells during muscle regeneration, but also has a broader mitotic activity on all myogenic cells. In view of the role of muscle regeneration in muscle diseases and of the perspectives offered by gene therapy via myoblasts, we strongly believe that our results open new opportunities in removing many of the clinical constraints associated with repair and cell transplantation.

Recovery of long-term denervated human muscles induced by electrical stimulation.

We investigated the restorative potential of intensive electrical stimulation in a patient with long‐standing quadriceps denervation. Stimulation started 18 months after injury. After 26 months, the thighs were visibly less wasted. Muscle cross‐sectional areas, measured by computerized tomography, increased from 36.0 cm2 to 57.9 cm2 (right) and from 36.1 cm2 to 52.4 cm2 (left). Knee torque had become sufficient to maintain standing without upper extremity support. Biopsies revealed evidence of both growth and regeneration of myofibers. The results suggest that electrical stimulation may offer a route to the future development of mobility aids in patients with lower motor neuron lesions. Muscle Nerve, 2004

Specific changes in skeletal muscle myosin heavy chain composition in cardiac failure: differences compared with disuse atrophy as assessed on microbiopsies by high resolution electrophoresis.

OBJECTIVE: In congestive heart failure (CHF) the skeletal muscle of the lower limbs develops a myopathy with atrophy and shift from the slow type to the fast type fibres. The aim was to test the hypothesis that this myopathy is specific and not simply related to detraining, by comparing patients with different degrees of CHF with patients with severe muscle atrophy due to disuse. DESIGN: Case-control study involving 50-150 micrograms needle biopsies of the gastrocnemius muscle. By an electrophoretic micromethod, the three isoforms of myosin heavy chains (MHC) were separated. PATIENTS: Five patients restricted to bed for more than one year because of stroke with disuse atrophy and normal ventricular function, and 19 with CHF were studied. There were seven age matched controls. MAIN OUTCOME MEASURES: The percentage of MHC1 (slow isoform), MHC2a (fast oxidative), and MHC2b (fast glycolytic) was determined by densitometric scan and correlated with indices of severity of cardiac failure. RESULTS: Ejection fraction was 42.5 (SD 15.2)% in CHF, 59.5 (1.0)% in disuse atrophy and 60.3 (1.4)% in controls (P < 0.001 v both). The degree of muscle atrophy as calculated by the body mass index/gastrocnemius cross sectional area, showed a profound degree of atrophy in patients with muscle disuse [0.94 (0.39)]. This was worse than in the controls [4.27 (0.16), P < 0.0005] and the CHF patients [2.60 (1.10), P < 0.005]. Atrophy in CHF patients was also greater than in controls (P < 0.005). MHC1 was lower in CHF than in disuse atrophy [51.83 (15.04) v 84.5 (17.04), P < 0.01] while MHC2b was higher [23.5 (7.4) v 7.25 (7.92), P < 0.001]. There was a similar trend for MHC2a [24.83 (15.01) v 8.25 (9.12), P < 0.05]. Within the CHF group there was a positive correlation between NYHA class and MHC2a (r = 0.47, P < 0.05) and MHC2b (r = 0.55, P < 0.01) and a negative correlation between NYHA class and MHC1 (r = -0.74, P < 0.001). Similarly, significant correlations were found for ejection fraction, diuretic consumption score, exercise test tolerance, and degree of muscle atrophy. CONCLUSIONS: The CHF myopathy appears to be specific and not related to detraining. The magnitude of MCH redistribution correlates with the severity of the disease. The electrophoretic micromethod used is very sensitive and reproducible. Biopsies are so well tolerated that can be repeated frequently, allowing thorough follow up.

Polymyositis, dermatomyositis and malignancy: A further intriguing link

The association between malignancy and autoimmune myositis has been largely described and confirmed by numerous epidemiological studies. The temporal relationship between the two pathologic conditions can vary: malignancy may occur before, at the same time or following the diagnosis of myositis. Beside these observations, the molecular mechanisms underlying this association are still unknown, even though it has been demonstrated a possible antigenic similarity between regenerating myoblasts and some cancer cell populations. To better identify peculiar histopathologic features common to cancer and myositis, we screened muscle biopsies from patients affected with polymyositis, dermatomyositis, myositis in association to cancer, and from patients affected with newly diagnosed cancer, but without myositis. Similarly to the histopatologic features that were observed in the muscle from myositis patients, especially in those with cancer associated myositis, in patients affected with malignancy at the clinical onset of disease we observed early sign of myopathy, characterized by internally nucleated and regenerating myofibers, most of them expressing the neural cell adhesion molecule. The hypothesis that in a particular subset of individuals genetically predisposed to autoimmunity, an initial subclinical tumor-induced myopathy may result in an autoimmune myositis, represents a further intriguing link behind the association of these two conditions.

Lifelong Physical Exercise Delays Age-Associated Skeletal Muscle Decline

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

Structural differentiation of skeletal muscle fibers in the absence of innervation in humans

The relative importance of muscle activity versus neurotrophic factors in the maintenance of muscle differentiation has been greatly debated. Muscle biopsies from spinal cord injury patients, who were trained with an innovative protocol of functional electrical stimulation (FES) for prolonged periods (2.4–9.3 years), offered the unique opportunity of studying the structural recovery of denervated fibers from severe atrophy under the sole influence of muscle activity. FES stimulation induced surprising recovery of muscle structure, mass, and force even in patients whose muscles had been denervated for prolonged periods before the beginning of FES training (up to 2 years) and had almost completely lost muscle-specific internal organization. Ninety percent (or more) of the fibers analyzed by electron microscopy showed a striking recovery of the ultrastructural organization of myofibrils and Ca2+-handling membrane systems. This functional/structural restoration follows a pattern that mimics some aspects of normal muscle differentiation. Most importantly, the recovery occurs in the complete absence of motor and sensory innervation and of nerve-derived trophic factors, that is, solely under the influence of muscle activity induced by electrical stimulation.

Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower motor neuron lesion

Background. Spinal cord injury causes muscle wasting and loss of function, which are especially severe after complete and permanent damage to lower motor neurons. In a previous cross-sectional study, long-standing denervated muscles were rescued by home-based functional electrical stimulation (h-bFES) training. Objective. To confirm results by a 2-year longitudinal prospective study of 25 patients with complete conus/cauda equina lesions. Methods. Denervated leg muscles were stimulated by h-bFES using a custom-designed stimulator and large surface electrodes. Muscle mass, force, and structure were determined before and after 2 years of h-bFES using computed tomography, measurements of knee torque during stimulation, and muscle biopsies analyzed by histology and electron microscopy. Results. Twenty of 25 patients completed the 2-year h-bFES program, which resulted in (a) a 35% cross-sectional increase in area of the quadriceps muscle from 28.2 ± 8.1 to 38.1 ± 12.7 cm 2 (P < .001), a 75% increase in mean diameter of muscle fibers from 16.6 ± 14.3 to 29.1 ± 23.3 μm (P < .001), and improvements of the ultrastructural organization of contractile material; and (b) a 1187% increase in force output during electrical stimulation from 0.8 ± 1.3 to 10.3 ± 8.1 N m (P < .001). The recovery of quadriceps force was sufficient to allow 25% of the subjects to perform FES-assisted stand-up exercises. Conclusions. Home-based FES of denervated muscle is an effective home therapy that results in rescue of muscle mass and tetanic contractility. Important immediate benefits for the patients are the improved cosmetic appearance of lower extremities and the enhanced cushioning effect for seating.

Maturation, dystrophic changes and the continuous production of fibers in skeletal muscle regenerating in the absence of nerve.

To investigate how much skeletal muscle depends on influences coming from peripheral nerve, we studied muscle regeneration induced by bupivacaine in the permanently denervated soleus muscle of adult rats. Using light and electron microscopy, we studied the extent of maturation attained by regenerated myofibers and their ability to regenerate again after repeated bupivacaine injury. Morphometric analyses showed that within the first two weeks after injury, the regenerated denervated fibers increased in diameter, matured and then became atrophic. By electron microscopy the morphological characteristics of mature fibers are evident, but final differentiation into adult fiber types is not attained. This is in keeping with previously reported biochemical results. A new phase of massive regeneration occurred when bupivacaine treatment was repeated, thus demonstrating that regenerated myofibers devoid of innervation keep their intrinsic ability to regenerate after injury. Spontaneous though scanty regeneration also occurred in denervated regenerated muscle at late stages, when atrophy and dystrophy of fibers became prominent. Concomitantly the relative number of myosatellite cells remained high. These morphological observations further support the hypothesis that fiber regeneration participates in the maintenance of chronically denervated muscles.