Katia Rossini

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

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.

Beneficial Effects on Skeletal Muscle of the Angiotensin II Type 1 Receptor Blocker Irbesartan in Experimental Heart Failure

BackgroundIn congestive heart failure (CHF), skeletal muscle shows increased expression of fast myosin heavy chains (MHC) and fibers, muscle atrophy, increased fatigability, and decreased endurance. Atrophy is secondary to myocyte apoptosis, which is probably triggered by tumor necrosis factor-&agr; (TNF&agr;). Angiotensin II receptors are thought to play a role in controlling apoptosis. We tested the hypothesis that angiotensin II receptor blockade could prevent skeletal muscle apoptosis in rats with CHF. Methods and ResultsCHF was induced by injecting 36 rats with 30 mg/kg monocrotaline. Ten additional animals were injected with saline and acted as controls. After 2 weeks, 18 of the 36 rats with CHF were treated with 7 mg · kg−1 · d−1 irbesartan through osmotic minipumps, and 10 of the 36 rats were treated with 2 mg · kg−1 · d−1 nifedipine in drinking water. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, MHC composition, myocyte apoptosis, Bcl-2, pro-caspase 3, and activated caspases 3 and 9 were determined, as were plasma levels of TNF&agr; and angiotensin II. Myocyte apoptosis and muscle atrophy were significantly decreased with irbesartan compared with untreated CHF rats. Irbesartan-treated rats had fewer cells labeled positively with terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling and fewer caspases; however, they also had increased Bcl-2 levels and muscle fiber cross-sectional areas. The MHC pattern in irbesartan-treated animals was similar to that in controls. Nifedipine animals behaved like the untreated CHF animals. Angiotensin II was increased 3- to 4-fold in all CHF rats (treated and untreated). TNF&agr; levels were decreased in irbesartan-treated rats but not in nifedipine-treated rats. ConclusionsAngiotensin II receptor blockade can protect from the development of apoptosis-dependent atrophy and from changes in MHCs. The reduction of TNF&agr; may play a role in this process.

Muscle biopsies show that FES of denervated muscles reverses human muscle degeneration from permanent spinal motoneuron lesion.

This paper presents biopsy analyses in support of the clinical evidence of muscle recovery induced by a new system of life-long functional-electrical-stimulation (FES) training in permanent spinal-motoneuron-denervated human muscle. Not earlier than 1 year after subjects experienced complete conus cauda lesion, their thigh muscles were electrically stimulated at home for several years with large skin surface electrodes and an expressly designed stimulator that delivered much longer impulses than those presently available for clinical use. The poor excitability of long-term denervated muscles was first improved by several months of twitch-contraction training. Then, the muscles were tetanically stimulated against progressively increased loads. Needle biopsies of vastus lateralis from long-term denervated subjects showed severe myofiber atrophy or lipodystrophy beginning 2 years after spinal cord injury (SCI). Muscle biopsies from a group of 3.6- to 13.5-year denervated subjects, who underwent 2.4 to 9.3 years of FES, show that this progressive training almost reverted long-term muscle atrophy/degeneration.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

Abstract This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2–24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

Activity–rest stimulation of latissimus dorsi for cardiomyoplasty: 1-year results in sheep

BACKGROUND In dynamic cardiomyoplasty electro-stimulation achieves full transformation of the latissimus dorsi (LD); therefore, its slowness limits the systolic support. Daily activity-rest could maintain partial transformation of the LD. METHODS Sheep LD were burst-stimulated either 10 or 24 hours/day. Before and 2, 4, 6, and 12 months after stimulation, LD power output, fatigue resistance, and tetanic fusion frequency were assessed. Latissimus dorsi were biopsied at 6 months, and sheep sacrificed at 12 months. RESULTS After 1 year of 10 hours/day stimulation LD was substantially conserved and contained large amounts of fast type myosin. From 2 months to 1 year of stimulation the power per muscle of the daily rested LD was greater than that of the left ventricle, being three to four times higher than in the 24-hour/day stimulation. CONCLUSIONS If extended to humans, these results could be the rationale for the need of a cardiomyostimulator, whose discontinuous activity could offer to patients the long-standing advantage of a faster and powerful muscle contraction.

Reconstruction of ablated rat rectus abdominis by muscle regeneration.

Skeletal muscle regeneration is a powerful, naturally occurring process of tissue reconstruction that follows myofiber damage secondary to myotoxic injury that does not normally affect the tissue circulation and scaffold. The ablated tissue, in traumatology and free muscle grafts, is frequently replaced by scars. The final outcome is poor even after in situ myoblast seeding of the harvested muscle. The goal of this study was to identify protocols to reconstruct muscle tissue, even in such adverse environments. The authors applied a step-by-step approach to identify factors favoring the survival of autologous satellite cells and, thus, muscle regeneration. In a rat model of full-thickness rectus abdominis muscle ablation, autologous myoblasts were isolated from the explanted rectus abdominis and seeded in a homologous acellular matrix immediately after wall reconstruction (group 1, five animals). In group 2 (five animals), the ablated rectus abdominis was autografted in situ. In a third group of five rats, Marcaine was injected into both the autograft and the surrounding abdominal wall muscle. Three weeks after surgery, serial cross-sections of the reconstructed abdominal wall were stained with hematoxylin and eosin or embryonic myosin antibody, a well-characterized molecular marker of early myogenesis in development and regeneration. Percentages of the patch area covered by regenerated myofibers were determined by morphometry. When autologous myoblasts were seeded in a homologous acellular matrix, the only myofibers observed to regenerate were those along the border of the patch. Autografting of the middle third of the rectus abdominis muscle similarly resulted in scar formation. The few muscle cells in the graft core were scanty myoblasts that could be detected only by monoclonal embryonic myosin antibody. Although negative for myofiber regeneration, the results in both cases confirmed the mechanical patency of the patches with regard to abdominal organ support. Myofibers were successfully regenerated in the graft by injecting Marcaine into both the autograft and the surrounding muscles. Three weeks after surgery, the patch was paved with young, centrally nucleated myofibers intermixed with young myofibers and myotubes expressing embryonic myosin. The difference in percentage of patch area covered by regenerated myofibers in group 3 (Marcaine injection around the patch, 81.6 +/- 3.0 percent) (mean +/- SD) versus either group 1 (Myoblast-seeded acellular patch, 18.0 +/- 3.0 percent) or group 2 (Autograft, 25.8 +/- 7.0 percent) was statistically significant on independent t test analysis (p < 0.0001). Even an acellular matrix showed some myofiber regeneration after surrounding muscles had been injected with Marcaine. This is the first successful evidence of muscle reconstruction after full-thickness ablation of the middle third of the rectus abdominis. Muscle regeneration seems to be the result of successive waves of migration of angioblasts and then satellite cell-derived myoblasts from the muscles surrounding the patch. The results strongly suggest that vascularization of the scaffold and successive coordinate proliferation of the seeded cells are required for myoblasts to be able to migrate into the patch and differentiate up to myofiber stage.

Chronic intermittent stimulation of the thyroarytenoid muscle maintains dynamic control of glottal adduction

Patients with laryngeal motor control disorders need improved dynamic glottal closure for speech and swallowing. To evaluate the functional outcome of intermittent chronic thyroarytenoid muscle stimulation in an animal model, 6 canines were implanted with bilateral Medtronic Xtrel systems containing Peterson‐type electrodes in the inferior and superior portions of the thyroarytenoid muscle. Stimulation was on one side only at 60 Hz, for 5 s on and 5 s off, over 8 h, 5 days per week, up to 8 months. Monthly videorecordings were done under anesthesia to measure the voltage threshold for detectable movement on each side, and vocal fold displacement and velocity during maximal stimulation of each side. Movement thresholds were lower in the inferior portion of the thyroarytenoid muscle (P ≤ 0.0005). Movement velocity was greater on the stimulated than on the nonstimulated side after 3 to 8 months (P = 0.039). No differences in the percentage distribution of different myosin heavy chain types were found between the stimulated and nonstimulated muscle samples. Sustained dynamic glottal adduction with no alteration in thyroarytenoid muscle function or fiber type was achieved with intermittent stimulation over 8 months. The results suggest that chronic intermittent thyroarytenoid stimulation has good potential for improving airway protection in dysphagia.

Inhibition of FasL sustains phagocytic cells and delays myogenesis in regenerating muscle fibers

Macrophage‐muscle cell interactions are complex, and the majority is unknown. The persistence of inflammatory cells in skeletal muscle could be critical for myofiber viability. In the present paper, we show that FasL plays a role in the resolution of muscle inflammation. We analyzed inflamed muscles of normal mice treated from day 3 to day 8 with a FasL inhibitor (Fas‐Ig) or with control Ig. Treated muscles were collected at 3, 5, and 10 days. The treatment with recombinant Fas‐Ig protein induced a severe persistence of inflammatory cells at 5 days (115,000±27,838 vs. 41,661±6848, p<0.01) and 10 days from injury (145,500±40,850 vs. 5000±1000, p<0.001). Myofiber regeneration was highly impaired (37±14 vs. 252±28, p<0.01). Apoptosis of phagocytic cells was absent during Fas‐Ig treatment (0.9±0.6 vs. 1300±150,p<0.0001), but apoptotic, mononucleated cells appeared at day 10, 2 days after the suspension of Fas‐Ig administration. The time course of FasL expression during muscle inflammation, at mRNA and protein level, reveals a peak during myoblast proliferation. The peak of FasL expression coincides with the peak of apoptosis of phagocytic cells. In situ hybridization shows the co‐expression of FasL and MyoD mRNA in mononucleated cells, i.e., myoblasts. Experiments on the myoblast cell culture confirmed the expression of FasL in myoblasts. The findings shown here indicate one of the pathways to control myoblast‐macrophage interaction and might be relevant for the control of inflammatory cells in muscle tissue. Perhaps altering FasL expression with recombinant proteins could ameliorate inflammation in degenerative myopathies and up‐regulate muscle regeneration.