Long-term administration of the TNF blocking drug Remicade (cV1q) to mdx mice reduces skeletal and cardiac muscle fibrosis, but negatively impacts cardiac function.

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in the gene encoding dystrophin (DYS). Tumor necrosis factor (TNF) has been implicated in the pathogenesis since short-term treatment of mdx mice with TNF blocking drugs proved beneficial; however, it is not clear whether long-term treatment will also improve long-term outcomes of fibrosis and cardiac health. In this investigation, short and long-term dosing studies were carried out using the TNF blocking drug Remicade and a variety of outcome measures were assessed. Here we show no demonstrable benefit to muscle strength or morphology with 10mg/kg or 20mg/kg Remicade; however, 3mg/kg produced positive strength benefits. Remicade treatment correlated with reductions in myostatin mRNA in the heart, and concomitant reductions in cardiac and skeletal fibrosis. Surprisingly, although Remicade treated mdx hearts were less fibrotic, reductions in LV mass and ejection fraction were also observed, and these changes coincided with reductions in AKT phosphorylation on threonine 308. Thus, TNF blockade benefits mdx skeletal muscle strength and fibrosis, but negatively impacts AKT activation, leading to deleterious changes to dystrophic heart function. These studies uncover a previously unknown relationship between TNF blockade and alteration of muscle growth signaling pathways.

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3.

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

Adsorption of atomic nitrogen and oxygen on [Formula: see text] surface: a density functional theory study.

The adsorption of atomic nitrogen and oxygen on the ([Formula: see text]) crystal face of zinc oxide (ZnO) was studied. Binding energies, workfunction changes, vibrational frequencies, charge density differences and electron localization functions were calculated. It was elucidated that atomic oxygen binds more strongly than nitrogen, with the most stable [Formula: see text] structure exhibiting a binding energy of -2.47 eV, indicating chemisorption onto the surface. Surface reconstructions were observed for the most stable minima of both atomic species. Positive workfunction changes were calculated for both adsorbed oxygen and nitrogen if the adsorbate interacted with zinc atoms. Negative workfunction changes were calculated when the adsorbate interacted with both surface oxygen and zinc atoms. Interactions between the adsorbate and the surface zinc atoms resulted in ionic-type bonding, whereas interactions with oxygen atoms were more likely to result in the formation of covalent-type bonding. The positive workfunction changes correlate with an experimentally observed increase in resistance of ZnO conductometric sensor devices.

Albuterol increases lean body mass in ambulatory boys with Duchenne or Becker muscular dystrophy.

BACKGROUND: Albuterol is a beta-2 agonist that has been demonstrated to increase muscle strength in studies in animals and humans. Based on a pilot study of extended-release albuterol Repetabs in children with dystrophinopathies, the authors conducted a randomized, double-blind, placebo-controlled study with a crossover design. METHODS: Fourteen boys with Duchenne or Becker muscular dystrophy, 6 to 11 years old, completed two treatment periods (albuterol and placebo), 12 weeks each, separated by a 12-week washout period. As the albuterol Repetab formulation was no longer available, an alternate extended release albuterol was used (Volmax, 12 mg per day). Outcome measurements included 1) lean body mass, 2) fat mass, 3) isometric knee extensor and flexor moments, 4) manual muscle testing, and 5) timed functional tests. RESULTS: Lean body mass was significantly higher for subjects following albuterol treatment compared to placebo treatment, while fat mass was significantly lower. No differences were found in isometric knee moments or manual muscle tests. Time to run/walk 30 feet was improved following albuterol. CONCLUSIONS: Short-term treatment with extended release albuterol may increase lean body mass, decrease fat mass, and improve functional measures in patients with dystrophinopathies. However, the significant change in strength of specific muscle groups found in the pilot study was not observed in the present study. These findings may be attributed to differences in the drug release and kinetics between Repetab and Volmax formulations as they affect the concentration of available beta-2 receptors on the muscle cell surface differently.

Pilot trial of albuterol in Duchenne and Becker muscular dystrophy.

The authors conducted a randomized, crossover, double-blind, placebo-controlled pilot study of albuterol in nine boys with dystrophinopathies. Primary outcomes were 1) isometric knee extensor and flexor strength; and 2) manual muscle testing (MMT). Isometric knee extensor strength and MMT scores were significantly higher in patients taking albuterol vs placebo. Therefore, 12-week treatment with extended-release albuterol may increase strength in patients with dystrophinopathies. A larger, double-blind, randomized study is necessary to confirm these results.

Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin.

Using both in vitro and in vivo approaches, numerous studies have provided evidence that mu-calpain is responsible for postmortem proteolysis. This paper reports the effect of overexpression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8) with a human calpastatin gene, whose expression was driven by the human skeletal muscle actin promoter, were killed along with control nontransgenic littermates (n = 5). Hind limbs were removed and stored at 4 degrees C, and muscle samples were dissected at 0, 1, 3, and 7 d postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater (P < 0.001) than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 d postmortem, approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (mu- and m-) expression was unaffected (P > 0.05) by the transgene as assessed by immunoreactivity at d 0. Over 7 d, 33% of at-death 80-kDa isoform immunoreactivity of mu-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of mu-calpain was slowed in transgenic mice. Desmin degradation was also inhibited (P < 0.05) in transgenics when compared to controls. Control mice lost 6, 78, and 91% of at-death native desmin at 1, 3, and 7 d postmortem, respectively; conversely, transgenic mice lost only 1, 3, and 17% at the same times. A similar trend was observed when examining the degradation of troponin-T. Interestingly, m-calpain seemed to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both mu- and m-calpain are active postmortem in normal murine skeletal muscle. In conclusion, a high level of expression of active calpastatin was achieved, which, by virtue of its inhibitory specificity, was determined to be directly responsible for a decrease in postmortem proteolysis.

Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation.

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.

A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice.

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.

Do immune cells promote the pathology of dystrophin-deficient myopathies?

Many features of dystrophin-deficient muscle pathology are not clearly related to the loss of mechanical support of the muscle membrane by dystrophin. In the present review, evidence that supports a role for the immune system in promoting the pathology of dystrophinopathy is presented. The findings summarized here indicate that specific, cellular immune responses by cytotoxic T-lymphocytes and helper T-lymphocytes contribute to muscle pathology in dystrophin-deficient muscle, and that removal of specific lymphoid cell populations can reduce muscle pathology. In addition, innate immune responses may also promote dystrophinopathies by the tremendous infiltration of myeloid cell populations into the dystrophic muscle. Loss of normal redox homeostasis by dystrophin-deficient muscle may increase its sensitivity to free radical-mediated damage by myeloid cells. Collectively, the observations presented here suggest that the contribution of the immune system to dystrophinopathies may be significant, and that therapeutic approaches based upon immune interventions may ameliorate the pathological progression of dystrophin deficiency.

Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.

Duchenne muscular dystrophy (DMD) and mdx mouse dystrophy result from mutations in the dystrophin gene. Although these mutations are primarily responsible for the defects that underlie the pathology of dystrophinopathies, other factors may contribute importantly to the pathology. In the present investigation, we tested whether T cells present in mdx muscles are activated and contribute significantly to the pathological process. Flow cytometric analyses showed a significantly higher frequency of activated CD44(high) T cells in the blood and muscle of mdx mice when compared to normal B10 mice. However, the frequency of activated T cells was not elevated in mdx lymph nodes, suggesting muscle-specific T cell activation. In vivo antibody-mediated depletions of CD4(+) T cells from mdx mice significantly reduced the amount of histologically discernible muscle pathology by 61% in mdx mice, while depletion of CD8(+) T cells resulted in a 75% decrease in pathology. Finally, adoptive transfer of mdx immune cells in combination with muscle extracts resulted in muscle pathology in healthy murine recipients. These results indicate that T cells promote the mdx pathology and suggest that immune-based therapies may provide benefit to DMD patients.

Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice.

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF-/mdx). Body mass and muscle mass of the TNF-/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.

Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors.

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

Calpains and muscular dystrophies.

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.

Nitric-oxide synthase is a mechanical signal transducer that modulates talin and vinculin expression.

Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

Influence of gender and socio-economic status on dietary patterns and nutrient intakes in 18-year-old Australians.

This study used two-day diet records to examine dietary behaviours in 504 Australian 18 year-olds in relation to gender, socio-economic status (SES) and national dietary guidelines. Fat intake exceeded 30% of energy in about 80% of subjects and was greater than 40% in about one-quarter. Saturated fat provided more than 10% of dietary energy in more than 90% of participants; less than 1% achieved a polyunsaturated to saturated fat ratio of at least one. The major food groups contributing to fat intake were convenience foods (32% in men, 28% in women) and meat (27% in men, 25% in women). Fibre intake was less than 30 g/day in 93% of women and 77% of men. Intakes of calcium, magnesium, potassium, and vitamins C and A, as a ratio of energy consumption, were greater in women than men, while sodium intake was significantly higher in men. Convenience foods were the greatest contributors to sodium intake (27% in men, 22% in women) followed by meat, bread, and soups and sauces. Greater consumption of cereals, fruit, vegetables and low-fat foods in young women of higher SES was reflected in their nutrient profile with higher intake of fibre and vitamin C and lower intake of fat. Men ate more cereals, meat and sugary foods and less fruit, vegetables and low-fat foods. Only 2.5% of men and 4.1% of women conformed with the health promotion message, widely publicised locally, to eat two fruits and five vegetables daily. Not eating breakfast was associated with lower calcium intake in men and women, and lower iron and fibre in take in women. Achieving behavioural changes in young adults must take into account differences in dietary behaviour related to gender and SES.

A controlled trial of health promotion programs in 11-year-olds using physical activity "enrichment" for higher risk children.

OBJECTIVE: To evaluate the short and long term benefits of a school and home based physical activity "enrichment" program for children at higher risk of cardiovascular disease as identified by cluster analysis. STUDY DESIGN: During two 10-week school terms, 800 11-year-olds took part in a randomized controlled trial with the standard physical activity and nutrition program in six schools, the standard program in a further seven schools but with the addition of physical activity enrichment for higher risk children in those schools, and no program in five control schools. Cluster analysis identifying the 29% or so highest risk children used systolic blood pressure, percent body fat, physical fitness, and blood cholesterol. RESULTS: Fitness improved significantly in program schools, particularly with enrichment in higher risk boys. Substantial improvements persisted 6 months later in girls from program schools. At "Enrichment" schools, cholesterol showed significant benefits in higher risk girls and, 6 months later, in both boys and higher risk girls. Sodium intake and, in girls, subscapular skinfolds were lower in "Enrichment" schools when the program ended, but not 6 months later. CONCLUSION: Two-semester health programs with physical activity enrichment for higher risk children can produce benefits sustained for at least 6 months. Improvements extend to lower risk children exposed indirectly to the enrichment. Attenuation of effects on diet and body composition in the longer-term suggest the need for on-going programs.

Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat.

High fat diets increase body fat stores. The following experiment was undertaken to determine whether the type of dietary fat could influence fat storage and whether voluntary exercise could prevent diet-induced obesity in mice fed high fat diets. Sixty-nine 6-wk-old female mice were fed one of three diets: low fat (11.5% of energy from fat), beef fat (40.8% of energy from fat) or canola oil (40.8% of energy from fat). In each diet group, 13 mice had free access to activity wheels in their cages (exercising), and the remaining 10 mice were housed in standard mouse cages (nonexercising). Body weight and body composition were measured before and after 8 wk of treatment. The nonexercising mice fed beef fat weighed more and had significantly more body fat (23.2 +/- 2.5 g/100 g body wt) than mice fed the low fat or canola oil diet (13.9 +/- 1.7 and 16.8 +/- 1.9 g/100 g body wt, respectively). Voluntary exercise did not affect lean body mass but did result in significantly lower body fat in all diet groups (beef, 12.6 +/- 0.9; low fat, 7.4 +/- 0.6; canola oil, 9.6 +/- 1.4 g/100 g body wt). The amount of body fat of mice fed the monounsaturated canola oil was significantly less than that of mice fed the beef fat diet, suggesting that the type of fat as well as the amount of fat influences body fat stores. Furthermore, voluntary exercise decreased body fat in all mice and prevented diet-induced obesity in mice fed diets high in fat.