An integrated approach in a case of facioscapulohumeral dystrophy

BACKGROUND: Muscle fatigue, weakness and atrophy are basilar clinical features that accompany facioscapulohumeral dystrophy (FSHD) the third most common muscular dystrophy.No therapy is available for FSHD. CASE PRESENTATION: We describe the effects of 6mo exercise therapy and nutritional supplementation in a 43-year-old woman severely affected by FSHD. CONCLUSION: A mixed exercise program combined with nutritional supplementation can be safely used with beneficial effects in selected patients with FSHD

Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging

The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy

Temperature Dependence of MgATP and MgADP Affinity of Fast and Slow Rat Myosin Isoforms: An in Vitro Motility Assay Approach

It has been suggested that the rate of ADP release from acto-myosin and the rate of acto-myosin dissociation by ATP may play different roles to define unloaded shortening velocity of slow and fast myosins and tha their role might change with temperature (Nytray et al. 2006; Iorga et al. 2007). In this study, the in vitro motility assay (IVMA) approach was used to study the effect of MgATP and MgADP on actin sliding velocity (Vf) on slow and fast skeletal myosin isoforms at different temperatures. The velocity of actin filaments sliding on pure slow (myosin 1) and pure fast (myosin 2B) myosin isoforms from the rat was determined in a range of [MgATP] (0.01-2mM) and in the presence or absence of 2mM MgADP. Experiments were performed at 20, 25 and 35 °C. The rate constants of ADP release and ATP binding to acto-myosin were calculated. The inhibition of Vf by MgADP was greater in slow then fast isoforms and the rate constant of ADP release was higher in fast than slow isoform. The results suggest that, in slow isoforms, the maximum velocity could be limited by the rate of ADP dissociation from actomyosin. The ADP inhibitory effect decreased and the rate constant of ADP release increased in each isoform with temperature accounting for the increase in Vf. No differences were found between isoforms and among temperatures in the rate constant of ATP binding. The possibility that the rate of acto-myosin dissociation induced by ATP could play a role in defining Vf in fast isoforms will be discussed

Actomyosin interaction at low ATP concentrations

In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP] = 2 mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged \u201crigor\u201d-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of \u394GATP involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative \u394GATP values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction

Novel Approach Applied to IVMA to Study the Modulation of the Actomyosin Interaction by MgATP In Fast Skeletal Muscle

In this study we used an “in vitro motility assay” (IVMA) approach to investigate the effect of the variation of [MgATP] in determining the number and the sliding velocity (Vf) of actin filaments moved by fast skeletal myosin. Vf was studied on type 2B HMM from rats at 25C°, 100mM ionic strength and at varius [MgATP], [MgADP] and [Pi]. We designed a new experimental set-up to perform experiments at lower ionic strength and in buffers that had no interference with the ionic environment, in order to mimic physiological condition. This set-up allowed a complete and chemical speciation of the solutions opening the possibility to perform an accurate thermodynamic study. Therefore, along with kinetic measurements also quantitative thermodynamics measurements were carried obtaining the ∈″G of MgATP hydrolysistaking into account pH and [Mg2+]. We correlate the thermodynamics property of the system to Vf and to the number of sliding actin filaments which were assessed by a purpose-designed software. Preliminary results indicate: a) no straightly correlation between values of ∈″GATPand the velocity of actin filaments, b) an increase in thenumberof sliding actin filaments at low [MgATP] and no changes when the ratio [MgATP] / [HMM] was kept constant c) a decrease in the velocity of actin filaments at [Pi]=30mM. More investigations are required to confirm the unexpected results that indicate a complex role of MgATP and its metabolites in the modulation of actomyosin interaction

Hindlimb unloading induces changes in actin sliding velocity of mouse pure myosin isoforms

Disuse muscle atrophy following hindlimb unloading (HU) can cause changes in unloaded shortening velocity (V0) at muscle fiber level (Zhong et al., 2006). The aim of this study was to clarify whether myosin motor function can be affected by HU, accounting for the observed changes in V0. The in vitro motility assay (IVMA) approach was used. In IVMA the properties of myosin motor could be investigated in the absence of other myofibrillar proteins by assessing actin sliding velocity (Vf) on isolated myosin. Vf is widely considered analogous to V0 as significant correlation between V0 and Vf was demonstrated (Canepari et al., 1999). Pure type 1 myosin from soleus (S) and type 2B myosin from gastrocnemius (GAS) muscle of HU and control (C) mice were analyzed. The results show that Vf on myosin 2B from HU was significantly slower (2.70 ± 0.32) then Vf on myosin 2B from C (4.11 ± 0.35), whereas no significant difference was found for myosin 1 (0.84 ± 0.17 vs. 0.89 ± 0.04). The results suggest that type 2B myosin motor could be functionally altered by HU. The effect could be related to a differentMLCisoform composition (Zhong et al., 2007) or a post-translational modification of myosin (Ramamurthy et al., 2003; Coirault et al., 2007). Preliminary analysis of MLC composition show no differences between HU and