Vasopressin (AVP), oxytocin (OT) and related peptides induce differentiation and hypertrophy in myogenic cells expressing the V1a-vasopressin receptor (V1aR) or the oxytocin receptor (OTR). Either receptor can transduce both ligand signals. Binding of AVP and OT to the V1aR the target cells activates phosphatidylinositol hydrolysis, which in turn releases Ca 2+ from internal stores. The AVP-dependent increase in cytosolic Ca 2+ induces the activation of calcium/calmodulin-dependent kinase (CaMK) and calcineurin signaling, two pathways required for the full expression of the differentiated phenotype. Here we investigate the role of V1aR in myogenesis and hypertrophy by ectopically restoring V1aR expression and function using the C2C12 cell line, which is an experimental model of satellite cells that do not respond to AVP treatment. Our results show that AVP treatment enhances myogenic differentiation in V1aR-transfected C2C12 cultures alone. Moreover, calcium imaging analyses performed in individual control and V1aR-transfected C2C12 cells demonstrated that the presence of V1aR is sufficient to make C2C12 cells responsive to neurohypophyseal hormones stimulation, as demonstrated by the rapid and sustained release of calcium from internal stores observed in V1aR-transfected cells. These data demonstrate that, despite the high levels of OTR expressed by C2C12 cells, both AVP and OT failed to stimulate the differentiation program, thereby indicating that the presence of V1aR is essential to mediate the effects of neurohypophyseal hormones on myogenic differentiation in C2C12 cells.
Aging is associated with a progressive loss of muscle mass and strength, a condition also known as sarcopenia of aging. Progressive muscle wasting has been observed in rodents, non human primates and humans. Caloric restriction is a very robust anti-aging intervention, whose effect may depend on level and duration. A severe caloric restriction was said to attenuate the loss of muscle mass and function with age. We have evaluated the effects of age and mild (one day of fasting every week) or severe caloric restriction (3 days fasting every week) on the weight of different muscles (extensor digitorum longus, soleus, tibialis anterior and diaphragm) of male rats. Our data show that sarcopenia of aging does not necessarily occur in all muscles (diaphragm appears to be immune) and that only a severe caloric restriction counteracts sarcopenia of aging. Functioning of diaphragm is related to energy consumption and may not decrease significantly per unit of weight with age. Thus our findings are in line with the hypothesis that both severe dietary anti-aging intervention and uninterrupted exercise in the physiological range, may counteract sarcopenia of aging.
Accelerated proteolysis through the ubiquitin-proteasome system has been recognized as a major contributor to muscle wasting, a serious complication frequently associated with a number of inflammatory disorders. Muscle expression of atrogin-1/MAFbx, a rate-limiting ubiquitin ligase for muscle wasting, is upregulated in various inflammatory conditions, and is considered a therapeutic target for muscle wasting. As one of the free radicals whose production is elevated in inflammatory conditions, nitric oxide (NO) is implicated in the pathogenesis of muscle wasting. To understand how inflammatory mediators upregulate atrogin-1/MAFbx expression, we tested the hypothesis that NO mediates the upregulation of atrogin-1/MAFbx expression. C2C12 myotubes were incubated with a cocktail comprised of TNF-α, interferon γ and lipopolysaccharide (LPS), which stimulated NO production and atrogin-1/MAFbx expression. Pre-incubation of the myotubes with nitric oxide synthase (NOS) inhibitor L-NAME or S-ethylisothiourea (SETU) blocked the stimulation of NO production by the cocktail. However, the stimulation of atrogin-1/MAFbx expression was not disrupted. Intraperitoneal administration of LPS to mice resulted in elevated atrogin-1/MAFbx expression in gastrocnemius muscle. But, pretreatment of the mice with L-NAME did not alter LPS stimulation of atrogin-1/MAFbx expression. Therefore, NO does not mediate upregulation of atrogin-1/MAFb expression by inflammatory mediators.
Glycogen storage disease type IV (GSD IV, or Andersen disease) is an autosomal recessive disorder due to the deficiency of 1,4-alpha-glucan branching enzyme (or glycogen branching enzyme, GBE1), resulting in an accumulation of amylopectin-like polysaccharide in muscle, liver, heart and central and peripheral nervous system. Typically, the presentation is in childhood with liver involvement up to cirrhosis. The neuromuscular form varies in onset (congenital, perinatal, juvenile and adult) and in severity. Congenital cases are rare, and fewer than 20 cases have been described and genetically determined so far. This form is characterized by polyhydramnios, neonatal hypotonia, and neuronal involvement; hepatopathy is uncommon, and the babies usually die between 4 weeks and 4 months of age. We report the case of an infant who presented severe hypotonia, dilatative cardiomyopathy, mild hepatopathy, and brain lateral ventricle haemorrhage, features consistent with the congenital form of GSD IV. He died at one month of life of cardiorespiratory failure. Muscle biopsy and heart and liver autoptic specimens showed many vacuoles filled with PAS-positive diastase-resistant materials. Electron-microscopic analysis showed mainly polyglucosan accumulations in all the tissues examined. Postmortem examination showed the presence of vacuolated neurons containing this abnormal polysaccharide. GBE1 biochemical activity was virtually absent in muscle and fibroblasts, and totally lacking in liver and heart as well as glycogen synthase activity. GBE1 gene sequence analysis revealed a novel homozygous nonsense mutation, p.E152X, in exon 4, correlating with the lack of enzyme activity and with the severe neonatal involvement. Our findings contribute to increasing the spectrum of mutation associated with congenital GSD IV.
We studied natural history and morphological features in 10 adult onset Acid Maltase Deficient (AMD) patients who were ambulant (age range 23-69 yrs), and 1 juvenile-onset AMD patient, who was wheelchair-bound and respirator-dependent (disease duration 36 yrs). Morphological features in muscle biopsy showed a vacuolar myopathy, there was Golgi apparatus proliferation within fibers, the autophagic vacuoles were positive at their periphery for caveolin-3 and dystrophin, documenting extensive protein turnover. We first clinically followed the patients periodically during the baseline period of the trial, by evaluating 5 limb-girdle muscles with the Medical Research Council Scale and monitored the performance of 4 functional tests (walking, climbing stairs, Gowers’ manoeuvre and rising from a chair). We then performed in 5 patients (4 adult-onset and 1 juvenile-onset) an open prospective clinical trial with a β2 agonist (albuterol) and pulsed branched-chain aminoacids (dose 500 ml 4% intravenous for 10 days subsequently every month) for six months. During this period, treated patients improved in functional score. In the 4 adultonset patients no side effects were found, while the only respirator-dependent juvenileonset patient, following an initial intravenous infusion of albuterol, presented signs of pulmonary oedema. He then well tolerated oral albuterol. The 5 treated patients continued oral albuterol treatment after the initial 6 months, for over 3 years. Our data suggest that in adult-onset AMD, the use of oral β2-agonist drug might be efficacious to antagonize muscle wasting and might be used as symptomatic therapy until enzyme replacement or aminoacid (leucine) therapy will be available.
Awaiting more successful strategies of gene therapy, longer viability of dystrophic muscle is a major goal in Duchenne Muscular Dystrophies (DMD). An alternative approach to insertion of mini-genes is targeted-exon-skipping therapy to obtain shrunk dystrophin by antisense oligonucleotides against splicing enhancer sequences, which in vitro selectively promote Becker-like Dystrophin mRNA and protein accumulation. Thus, it is time to test in vivo long-term efficacy and toxicity of oligos against the most clinically-relevant exons of human dystrophin gene. Pregnant mothers and littermates could be treated and the latter studied from two weeks after birth. Biopsies of leg muscles at onethreeand six-month will be studied after inducing muscle damage by exposing mice to spontaneous run in wheel-cage. At one-year-age respiratory muscles (diaphragm) will be also studied. Beside effects of oligo treatments on dystrophin expression at protein level, micromethods allow to quantify muscle damage/regeneration on thin needle biopsies, small enough to be performed on muscle of mice. Relevance to DMD therapy is obvious. On the light of potential risks of viral-gene therapy, major drawback of the antisense approach (i.e., lifelong frequent administrations) represents a major advantage in safety, since treatments could be temporary discontinued in case of side-effects. We are confident to pave the way to human trials by showing that the approach is safe, muscle-selective and effective, if DMD TEST will increase resistance to exercise-induced muscle injury and therefore substantially prolong viability of the dystrophic muscle.
Abnormalities of mitochondria have been reported in Myotonic Dystrophy (MD). Adrenergic activation is one of the major factors influencing exercise lactate production in healthy subjects. In order to assess the role of such activation in MD, we compared blood catecholamine levels to those of lactate during an incremental bicycle exercise in 9 MD patients and 6 controls. The lactate values reached significantly higher levels in patients than in controls at rest (2.91 ± 0.58 vs 1.44 ± 1.14 mmol/l, p<0.01, in average), at the anaerobic threshold (4.43 ± 1.11 vs 2.62 ± 0.73 mmol/l, p<0.05), at exercise peak (6.64 ± 1.34 vs 3.90 ± 0.99 mmol/l, p< 0.05) and at recovery (3.21 ± 0.94 vs 1.91 ± 0.19, p<0.01). Furthermore, the anaerobic lactate threshold (LT) in MD was acquired at lower workload (mostly in a range between 30% and 50% of the predicted normal maximal power output) compared to controls (6070%), this suggesting an early activation of the anaerobic metabolism in MD patients. On the contrary, catecholamine levels were not significantly different between patients and controls. These results indicate that abnormal lactate production in MD is independent of the adrenergic response to exercise and suggest a direct involvement of skeletal muscle oxidative metabolism in MD.
Following denervation, skeletal muscle undergoes rapid loss in both mass and contractile force, with an accompanying series of changes in structure, biochemistry and physiology. Morphologic features of the long-term denervated muscle suggest that the original fibers are lost and those seen are the results of repeated cycles of cell death and regeneration. Markers of myogenic events in adult muscles are activated satellite cells, presence of embryonic myosin and myogenic transcription factors. Electrical stimulation of permanent denervated muscle increases the mean size of the myofibers, maintains the sarcomeres and possibly prevents apoptosis/necrosis and secondary degeneration. We here describe histological, immunohistochemical and molecular methods, which identify markers of damage, regeneration, and repair on a muscle sample of just a few milligrams. A suitable source is a small biopsy, whose cryostat sections allow fiber typing and histopathology by immunochemical analyses. A few additional serial cryostat sections are used for molecular analyses. By light morphometry percent fat and/or interstitial tissue area, and fiber size distribution are determined. Myosin ATPases and immunohistochemistry are used to quantify fiber types, including early or late regenerated myofibers. By electron microscopy we identify abnormalities of sarcomeric organization, nuclear distribution, mitochondrial content, and activated satellite cells. Myosin heavy chain profiles are indicators of regenerative events and of muscle adaptation to changing demands. Total protein content, myosin: total protein and myosin: actin ratios are determined by SDS-PAGE to reliably complement or substitute morphometry in quantifying muscle trophism in skeletal muscle biopsies. Taken together the results of morphometry and molecular analyses solidly demonstrate that two-year-long FES substantially reverses the severe atrophy expected in four-year denervated human muscles, and that generative/regenerative myogenic events significantly contribute to the functional recovery of long-term paralyzed human muscles.
Abtract To assess, in vivo, muscle oxidative efficiency in older people we studied haematic lactate in blood samples obtained from 34 older and 10 young subjects before and after an incremental exercise performed on a treadmill, in order to identify a possible defect as a factor determining motor performance impairment. The exercise protocol consisted of 11 steps of 2 minutes of duration, at a constant speed of 3 km/h; the grade was 0 at the beginning and was incremented of 2.5% each step. Lactate was evaluated at rest and during recovery (at 1’, 5’, 10’ and 30’ after the end of the exercise). We found that lactate resting values didn’t show a significant difference between older and young people. During recovery, older people showed significantly higher values at any time. The analysis of results permitted to distinguish a subgroup of 11 out of 34 subjects with a behaviour similar to young’s one. In conclusion the abnormal lactate increase observed in this study points out a reduced oxidative muscle function in older people. Interestingly, the test employed allowed to identify two subgroups: with normal levels and with altered levels of lactate. The latter could due to a different individual mt-DNA damage susceptibility.
A bstract Expression o f polym orphic myofibrillar and sarcoplasm ic proteins w as investigated in the fish Scophthalm us maximus (L.) undergoing m etam orphosis. A range of electrophoretic techniques w as used to m onitor sequential synthesis o f isoforms from hatching to the adult stage. Two isoforms (larval and adult) o f m yosin light chain LC2 and troponin-I were suc cessively detected during turbot growth, in addition to variations in the peptide com position o f m yosin heavy chains. Two isoform s of troponin-T also appeared sequentially, but the first to make its appearance w as not detected until the juvenile stage. The com position o f alkali light chains, actin, tropom yosin, and troponin-C did no t seem to change as the fish progressed through the different stages. Parvalbum in isoform s were isolated and their phy sico-chemical param eters defined. As in the other fish exam ined so far, there appeared a succession o f larval (PA Ila and PA lib ) and adult (PA V) parvalbum in isoform s through the life o f the fish. A ll these biochemical changes occurred gradually in the course of turbot development, and did no t appear particularly related to m etam orphosis but rather to phy siological needs o f the different growth stages.
Hereditary spastic paraplegia (HSP) is a group of familial neurodegenerative disorders characterized by progressive lower limb spasticity and weakness due to degeneration of corticospinal axons. These disorders are classified both genetically, according to the mode of inheritance, and clinically, as pure and complicated forms. Recently the discovery that paraplegin, the defective protein in autosomal recessive HSP-SPG7, localizes in mitochondria allowed to foster the hypothesis that some mitochondrial dysfunctions can play a pathogenic role in HSP. The aim of our study was to indirectly evaluate oxidative metabolism in contracting mu scle, by assessing the anaerobic lactate threshold in 7 patients (5 M and 2 F, mean age 48.0±13.9 yrs) affected by HSP, both autosomal dominant or sporadic, during an incremental bicycle exercise. Analysis of venous lactate curve showed that lactate levels were significantly higher than in controls (peak normalised lactate: 378.8 vs. 271%, p<0.01). Furthermore, an early threshold of lactate was detected only in HSP patients. Even if other factors such as chronic spasticity or muscle deconditioning have to be taken into account in the interpretation of our data, these results suggest possible occurrence of mitochondrial involvement in skeletal muscle of HSP patients.
In Dynamic Cardiomyoplasty after stimulation by the standard clinical Protocol Latissimus Dorsi (LD) is highly fatigue-resistant, but shows undesirable dynamic characteristics. The conditioned LD could show more than fivefold reduction in shortening velocity and peak power. To obtain fatigue-resistance while preserving muscle force and velocity, we introduced the concept of daily activity-rest stimulation. LD wrap is allowed to rest during periods of hours (e.g., while the patient is asleep).Based on experimental results obtained with burst-intermittent stimulation in animals, we implemented in a small series of patients a light regime of LD activity-rest stimulation (cardiac-rate/based demand stimulation). To determine LD contractile characteristics we developed a new noninvasive diagnostic tool (LD wrap "mechanogram"). By mechanogram and echo Doppler imaging we determine: 1) optimal synchronization delay between the contraction of the cardiac events and LD wrap; and 2) the dynamic contractile characteristics of the LD flap based on analysis of tetanic fusion frequency (TFF).The extent of fast-to-slow transformation of contractile characteristics of the LD wrap is related to the stimulation protocols used. After Demand Dynamic Cardiomyoplasty in the eight patients who achieved at least 6-month follow-up (mean 14+/-3 months), there are no deaths. Quality of life is substantially improved with significant reduction of heart failure symptoms. In the subset of patients in which light stimulation started with the muscle conditioning and Demand Dynamic Cardiomyoplasty was introduced earlier than one-year after surgery, exercise capacity increases up to two-year post Demand Dynamic Cardiomyoplasty (VO2 max: pre-op 12.3+/-0.7 v.s. 16.6+/-1.7 two-year post-Demand Dynamic Cardiomyoplasty, p = 0.05).In conclusion, Demand Dynamic Cardiomyoplasty is safe, well tolerated, and by maintaining an intermediate fast-to-slow LD wrap conversion provides up to two-year post-operation excellent clinical results.
Vascular deafferentation of the Latissimus Dorsi Muscle (LDM) for dynamic cardiomyoplasty is an important cause of ischemia in the distal portion of the muscle with degeneration of muscle fibres and consequent reduction in the effectiveness of the surgery. Experimental studies suggest that a vascular delay procedure and a 10-day delay adaptation significantly improve the distal perfusion and function of the LDM flap for cardiomyoplasty. We here report a comparative histological and ultrastructural study of biopsies from unconditioned LDM flaps of two patients, taken 30 days after vascular deafferentation and dynamic cardiomyoplasty, and 10 days after a vascular delay procedure, respectively. In the first case, in the distal part of LDM we found muscle atrophy and degeneration, necrosis of capillaries, with swelling of endothelial cells and disruption of cytoplasmic organelles, and capillary neoangiogenesis. In the second patient, the distal LDM after vascular delay procedure show minimal muscle fibre degeneration, dilatation of capillaries with endothelial cell preservation, and scarce capillary neoangiogenesis. The present results seem to confirm that a vascular delay procedure on LDM for use with cardiomyoplasty may improve the LDM perfusion and function, inducing moderate morphological changes in muscle fibres and vasculature in comparison to the deafferentated LDM.
