Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Single-fiber electromyography of the laryngeal muscles.

Single-fiber electromyography (SFEMG) is useful in the evaluation of disorders of neuromuscular transmission and the assessment of motor unit morphology. Standard EMG techniques are used routinely in the evaluation of laryngeal dysfunction, but the feasibility of laryngeal SFEMG has not been established. We, therefore, performed laryngeal SFEMG in 10 normal individuals to demonstrate the feasibility of the technique and generate preliminary normative data. We also studied 2 patients with amyotrophic lateral sclerosis and 1 patient previously treated with botulinum toxin for comparative purposes.

Gene-mediated restoration of normal myofiber elasticity in dystrophic muscles.

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

Diet and gene interactions influence the skeletal response to polyunsaturated fatty acids.

Diets rich in omega-3s have been thought to prevent both obesity and osteoporosis. However, conflicting findings are reported, probably as a result of gene by nutritional interactions. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear receptor that improves insulin sensitivity but causes weight gain and bone loss. Fish oil is a natural agonist for PPARγ and thus may exert its actions through the PPARγ pathway. We examined the role of PPARγ in body composition changes induced by a fish or safflower oil diet using two strains of C57BL/6J (B6); i.e. B6.C3H-6T (6T) congenic mice created by backcrossing a small locus on Chr 6 from C3H carrying 'gain of function' polymorphisms in the Pparγ gene onto a B6 background, and C57BL/6J mice. After 9months of feeding both diets to female mice, body weight, percent fat and leptin levels were less in mice fed the fish oil vs those fed safflower oil, independent of genotype. At the skeletal level, fish oil preserved vertebral bone mineral density (BMD) and microstructure in B6 but not in 6T mice. Moreover, fish oil consumption was associated with an increase in bone marrow adiposity and a decrease in BMD, cortical thickness, ultimate force and plastic energy in femur of the 6T but not the B6 mice. These effects paralleled an increase in adipogenic inflammatory and resorption markers in 6T but not B6. Thus, compared to safflower oil, fish oil (high ratio omega-3/-6) prevents weight gain, bone loss, and changes in trabecular microarchitecture in the spine with age. These beneficial effects are absent in mice with polymorphisms in the Pparγ gene (6T), supporting the tenet that the actions of n-3 fatty acids on bone microstructure are likely to be genotype dependent. Thus caution must be used in interpreting dietary intervention trials with skeletal endpoints in mice and in humans.

Caloric restriction induces energy-sparing alterations in skeletal muscle contraction, fiber composition and local thyroid hormone metabolism that persist during catch-up fat upon refeeding.

Weight regain after caloric restriction results in accelerated fat storage in adipose tissue. This catch-up fat phenomenon is postulated to result partly from suppressed skeletal muscle thermogenesis, but the underlying mechanisms are elusive. We investigated whether the reduced rate of skeletal muscle contraction-relaxation cycle that occurs after caloric restriction persists during weight recovery and could contribute to catch-up fat. Using a rat model of semistarvation-refeeding, in which fat recovery is driven by suppressed thermogenesis, we show that contraction and relaxation of leg muscles are slower after both semistarvation and refeeding. These effects are associated with (i) higher expression of muscle deiodinase type 3 (DIO3), which inactivates tri-iodothyronine (T3), and lower expression of T3-activating enzyme, deiodinase type 2 (DIO2), (ii) slower net formation of T3 from its T4 precursor in muscles, and (iii) accumulation of slow fibers at the expense of fast fibers. These semistarvation-induced changes persisted during recovery and correlated with impaired expression of transcription factors involved in slow-twitch muscle development. We conclude that diminished muscle thermogenesis following caloric restriction results from reduced muscle T3 levels, alteration in muscle-specific transcription factors, and fast-to-slow fiber shift causing slower contractility. These energy-sparing effects persist during weight recovery and contribute to catch-up fat.

Effect of aerobic exercise on skeletal muscle mitochondrial content and function in older adults

Regular aerobic exercise training, which is touted as a way to ameliorate metabolic diseases, increases aerobic capacity. Aerobic capacity usually declines with advanced age. The decline in aerobic capacity is typically associated by a decrease in the quality of skeletal muscle. At the molecular level, this decreased quality comes in part from perturbations in skeletal muscle mitochondria. Of particular is a decrease in the total amount of mitochondria that occupy the skeletal muscle volume. What is not well established is if this decrease in mitochondrial content is due to inactive lifestyle or the process of aging. Herein, the work of the thesis shows a clear connection between mitochondrial content and aerobic capacity. This indicates that active individuals with higher VChmax levels also contain higher volumes of mitochondria inside their muscle as opposed to sedentary counterparts who have lower levels of mitochondrial content. Upon taking these previously sedentary individuals and entering them into an aerobic exercise intervention, they are able to recover their mitochondrial content as well as function to similar levels of lifelong athletes of the same age. Furthermore, the results of this thesis show that mitochondrial content and function also correlate with exercise efficiency. If one is more efficient, he/she is able to expend less energy for a similar power output. Furthermore, individuals who increase in efficiency also increase in the ability to oxidize and utilize fat during pro-longed exercise. This increased reliance on fat after the intervention is associated with an increased amount of mitochondria, particularly in the intermyofibrillar region of skeletal muscle. Therefore, elderly adults who were once sedentary were able to recover mitochondrial content and function and are able to reap other health benefits from regular aerobic exercise training. Aging per se does not seem to be the culprit that will lead to metabolic diseases but rather it seems to be a lack of physical activity. -- Un entraînement sportif d'endurance, connu pour réduire le risque de développer des maladies métaboliques, augmente notre capacité aérobie. La capacité aérobie diminue généralement avec l'âge. Ce déclin est typiquement associé d'une diminution de la qualité du muscle squelettique. Au niveau moléculaire, cette diminution est due à des perturbations dans les mitochondries du muscle squelettique,, ce qui conduit à une diminution de la quantité totale des mitochondries présentes dans le muscle squelettique. Il n'a pas encore été établi si cette diminution de la teneur mitochondriale est due à un mode de vie sédentaire ou au processus du vieillissement. Ce travail de thèse montre un lien clair entre le contenu mitochondrial et la capacité aérobie. Il indique que des personnes âgées actives, avec des niveaux de V02max plus élevés, possèdent également un volume plus élevé de mitochondries dans leurs muscles en comparaison à leurs homologues sédentaires. En prenant des individus sédentaires et leur faisant pratiquer une activité physique aérobie, il est possible d'accroître leur contenu de même que leur fonction mitochondriale à des niveaux similaires à ceux d'athlètes du même âge ayant pratiqué une activité physique tout au long de leur vie. De plus, les résultats de ce travail démontrent que le contenu et la fonction mitochondriale sont en corrélation avec l'efficiscience lors d'exercice physique. En agumentant l'effiscience, les personnes sont alors capables de dépenser moins d'énergie pour une puissance d'exercice similaire. Donc, un volume mitochondrial accru dans le muscle squelettique, associé à une fonction mitochondriale améliorée, est associté à une augmentation de l'effiscience. En outre, les personnes qui augmentent leur effiscience, augmentent aussi leur capacité à oxyder les graisses durant l'exercice prolongé. Une augmentation du recours au graisses après l'intervention est associée à une quantité accrue de mitochondries, en particulier dans la région inter-myofibrillaire du muscle squelettique. Par conséquent, les personnes âgées autrefois sédentaires sont en mesure de récupérer leur contenu et leur fonction mitochondriale ainsi que d'autres avantages pour la santé grâce à un entraînement aérobie régulier. Le vieillissement en soi ne semble donc pas être le coupable conduisant aux maladies métaboliques qui semblent plutôt être lié à un manque d'activité physique.

β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline/β-agonists influence muscle performance during exercise/stress in humans.

Application of intravoxel incoherent motion perfusion imaging to shoulder muscles after a lift-off test of varying duration.

Intravoxel incoherent motion (IVIM) MRI is a method to extract microvascular blood flow information out of diffusion-weighted images acquired at multiple b-values. We hypothesized that IVIM can identify the muscles selectively involved in a specific task, by measuring changes in activity-induced local muscular perfusion after exercise. We tested this hypothesis using a widely used clinical maneuver, the lift-off test, which is known to assess specifically the subscapularis muscle functional integrity. Twelve shoulders from six healthy male volunteers were imaged at 3 T, at rest, as well as after a lift-off test hold against resistance for 30 s, 1 and 2 min respectively, in three independent sessions. IVIM parameters, consisting of perfusion fraction (f), diffusion coefficient (D), pseudo-diffusion coefficient D* and blood flow-related fD*, were estimated within outlined muscles of the rotator cuff and the deltoid bundles. The mean values at rest and after the lift-off tests were compared in each muscle using a one-way ANOVA. A statistically significant increase in fD* was measured in the subscapularis, after a lift-off test of any duration, as well as in D. A fD* increase was the most marked (30 s, +103%; 1 min, +130%; 2 min, +156%) and was gradual with the duration of the test (in 10(-3) mm(2) /s: rest, 1.41 ± 0.50; 30 s, 2.86 ± 1.17; 1 min, 3.23 ± 1.22; 2 min, 3.60 ± 1.21). A significant increase in fD* and D was also visible in the posterior bundle of the deltoid. No significant change was consistently visible in the other investigated muscles of the rotator cuff and the other bundles of the deltoid. In conclusion, IVIM fD* allows the demonstration of a task-related microvascular perfusion increase after a specific task and suggests a direct relationship between microvascular perfusion and the duration of the effort. It is a promising method to investigate non-invasively skeletal muscle physiology and clinical perfusion-related muscular disorders.

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity

Metabolic origin of insulin resistance in obesity with and without type 2 (non-insulin-dependent) diabetes mellitus.

A metabolic hypothesis is presented for insulin resistance in obesity, in the presence or absence of Type 2 (non-insulin-dependent) diabetes mellitus. It is based on physiological mechanisms including a series of negative feed-back mechanisms, with the inhibition of the function of the glycogen cycle in skeletal muscle as a consequence of decreased glucose utilization resulting from increased lipid oxidation in the obese. It considers the inhibition of glycogen synthase activity together with inhibition of glucose storage and impaired glucose tolerance. The prolonged duration of increased lipid oxidation, considered as the initial cause, may lead to Type 2 diabetes. This hypothesis is compatible with others based on the inhibition of insulin receptor kinase and of glucose transporter activities.

Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.