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).

Beneficial effects on skeletal muscle of the angiotensin II type 1 receptor blocker irbesartan in experimental heart failure.

BACKGROUND: In 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-alpha (TNFalpha). 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 RESULTS: CHF 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 TNFalpha 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). TNFalpha levels were decreased in irbesartan-treated rats but not in nifedipine-treated rats. CONCLUSIONS: Angiotensin II receptor blockade can protect from the development of apoptosis-dependent atrophy and from changes in MHCS: The reduction of TNFalpha may play a role in this process.

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.

Caspase 3 expression correlates with skeletal muscle apoptosis in Duchenne and facioscapulo human muscular dystrophy. A potential target for pharmacological treatment?

Apoptosis was detected in different muscular diseases, including severe dystrophin deficiency, but apoptotic mechanisms are not completely described in adult skeletal muscle. Studying patients affected by Duchenne muscular dystrophy (DMD) and by facio-scapulo-humeral dystrophy (FSHD) we showed an increase of apoptotic myonuclei, bax, and bcl-2-positive myofibers. Positive correlation was detected between apoptotic nuclei and bax expression (p < 0.01). Expression of caspases was analyzed by RNase protection. Caspase transcript was not detected in normal skeletal muscles. DMD muscles expressed caspase 8, 3, 5, 2, 7 and Granzyme B mRNAs. Low levels of caspase 6, 3, and Granzyme B transcripts were detected in FSHD patients. Tissue levels of caspase 3 protein significantly correlated with apoptotic myonuclei (p < 0.05) and with bax expression (p < 0.01). In all DMD cases the activity of caspase 3 was increased, while the FSHD samples were heterogeneous. These data indicate that human skeletal muscle fibers. during the dystrophic process, modulate the expression of caspases and that caspase 3 is involved in myofiber cell death. opening new perspective in the pharmacological treatments of muscular dystrophies, such as the use of caspase inhibitors.

Nerve control of type 2A MHC isoform expression in regenerating slow skeletal muscle.

Bupivacaine-induced regeneration was studied in rat soleus muscle under several conditions, with the focus on type 2A and type 1 myosin heavy chain (MHC) isoform expression. In denervated muscles, type 1 was absent, whereas type 2A was widely expressed, a pattern of regeneration which appeared to be independent of fibrillation activity of the muscle. Both type 1 and type 2A isoforms were absent in muscles regenerated during tetrodotoxin (TTX) block of impulse conduction in the sciatic nerve, but type 2A was still present when the TTX block was associated with the vinblastine block of axoplasmic flow; vinblastine block alone caused the coexpression of type 1 and type 2A isoforms in the majority of fibers. These results suggest that axoplasmic flow carries some chemical factor that inhibits 2A MHC isoform expression. The results are also of clinical interest, contributing to the understanding of factors controlling muscle differentiation and adaptation.

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

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 (beta-dystroglycan, alpha-sarcoglycan, and gamma-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.

The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases.

Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen-antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B' and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.

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.

Reimplante imediato de incisivo superior de rato após remoçäo do ligamento periodontal cementário e alveolar / The immediate dental replantation of the upper incisor rat after remotion of the cementary and alveolar periodontal ligament

Foram empregados 24 ratos machos e sob anestesia geral, dos quais o incisivo superior direito foi extraído, sendo entäo todos os animais dividos em dois grupos de 12, que receberam os seguintes procedimentos: no grupo 1 (controle), o incisivo, após a extraçäo, foi mantido durante três minutos sobre gaze umedecida em soro fisiológico e, a seguir, reimplantado em seu respectivo alvéolo. No grupo II (tratado), o incisivo, logo após a extraçäo, teve o seu ligamento periodontal aderido ao cemento, removido através de raspagem com lâmina de bisturi n§ 11. A seguir, utilizando uma cureta adaptada, o ligamento periodontal remanescente junto à parede óssea alveolar foi removido através de curetagem cuidadosa. Logo após, o dente foi reimplantado em seu alvéolo. Seis animais de cada grupo foram sacrificados aos 10 e 40 dias pós-reimplante. As peças obtidas, após processamento laboratorial de rotina, foram incluídas em parafina para possibilitar a microtomia. Os cortes semi-seriados corados em hematoxilina e eosina para estudo histomorfológico. Os resultados obtidos mostram que nos reimplantes sem o ligamento periodontal cementário e alveolar, ocorreu discreta reabsorçäo cemento-dentinária. Näo foi observada, ainda, anquilose alvéolo-dental, dentro dos períodos analisados no presente trabalho

Human satellite cell proliferation in vitro is regulated by autocrine secretion of IL-6 stimulated by a soluble factor(s) released by activated monocytes.

We previously showed that macrophages, besides their scavenger role, selectively induce rat myoblast proliferation in vitro by releasing soluble factors. In this paper we demonstrate a relationship between human-activated monocytes and increased human myoblast proliferation due to IL-6 autocrine secretion by satellite cells. Indeed in the supernatants of muscle cultures treated with activated monocyte-conditioned medium we show by means of an ELISA quantitation a higher autocrine secretion of IL-6 associated with increased myoblast proliferation. This suggests that a growth factor(s) secreted by activated monocytes stimulates IL-6 production by myoblasts and then regulates proliferation of satellite cells.

Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.

High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins.

In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.