Interuniversitary Institute of Myology

Italy

Interuniversitary Institute of Myology

Italy
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Protasi F.,Interuniversitary Institute of Myology | Protasi F.,University of Chieti Pescara | Paolini C.,Interuniversitary Institute of Myology | Paolini C.,University of Chieti Pescara | And 7 more authors.
Journal of Muscle Research and Cell Motility | Year: 2011

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca 2+ binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca 2+ during excitation-contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca 2+ release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca 2+ re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca 2+ depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders. © 2011 Springer Science+Business Media B.V.


PubMed | University of California at San Diego, Venetian Institute of Molecular Medicine, University of Chieti Pescara, University of Siena and University of Padua
Type: Journal Article | Journal: American journal of physiology. Cell physiology | Year: 2016

We recently reported that skeletal muscle fibers of obscurin knockout (KO) mice present altered distribution of ankyrin B (ankB), disorganization of the subsarcolemmal microtubules, and reduced localization of dystrophin at costameres. In addition, these mice have impaired running endurance and increased exercise-induced sarcolemmal damage compared with wild-type animals. Here, we report results from a combined approach of physiological, morphological, and structural studies in which we further characterize the skeletal muscles of obscurin KO mice. A detailed examination of exercise performance, using different running protocols, revealed that the reduced endurance of obscurin KO animals on the treadmill depends on exercise intensity and age. Indeed, a mild running protocol did not evidence significant differences between control and obscurin KO mice, whereas comparison of running abilities of 2-, 6-, and 11-mo-old mice exercised at exhaustion revealed a progressive age-dependent reduction of the exercise tolerance in KO mice. Histological analysis indicated that heavy exercise induced leukocyte infiltration, fibrotic connective tissue deposition, and hypercontractures in the diaphragm of KO mice. On the same line, electron microscopy revealed that, in the diaphragm of exercised obscurin KO mice, but not in the hindlimb muscles, both M-line and H-zone of sarcomeres appeared wavy and less defined. Altogether, these results suggest that obscurin is required for the maintenance of morphological and ultrastructural integrity of skeletal muscle fibers against damage induced by intense mechanical stress and point to the diaphragm as the skeletal muscle most severely affected in obscurin-deficient mice.


de Stefanis D.,University of Turin | Mastrocola R.,University of Turin | Nigro D.,University of Turin | Costelli P.,University of Turin | And 2 more authors.
European Journal of Nutrition | Year: 2015

Purpose: In recent years, the increasing consumption of soft drinks containing high-fructose corn syrup or sucrose has caused a rise in fructose intake, which has been related to the epidemic of metabolic diseases. As fructose and glucose intake varies in parallel, it is still unclear what the effects of the increased consumption of the two single sugars are. In the present study, the impact of chronic consumption of glucose or fructose on skeletal muscle of healthy mice was investigated. Methods: C57BL/6J male mice received water (C), 15 % fructose (ChF) or 15 % glucose (ChG) to drink for up to 7 months. Lipid metabolism and markers of inflammation and autophagy were assessed in gastrocnemius muscle. Results: Increased body weight and gastrocnemius muscle mass, as well as circulating glucose, insulin, and lipid plasma levels were observed in sugar-drinking mice. Although triglycerides increased in the gastrocnemius muscle of both ChF and ChG mice (+32 and +26 %, vs C, respectively), intramyocellular lipids accumulated to a significantly greater extent in ChF than in ChG animals (ChF +10 % vs ChG). Such perturbations were associated with increased muscle interleukin-6 levels (threefold of C) and with the activation of autophagy, as demonstrated by the overexpression of LC3B-II (ChF, threefold and ChG, twofold of C) and beclin-1 (ChF, sevenfold and ChG, tenfold of C). Conclusions: The present results suggest that intramyocellular lipids and the pro-inflammatory signaling could contribute to the onset of insulin resistance and lead to the induction of autophagy, which could be an adaptive response to lipotoxicity. © 2015 Springer-Verlag Berlin Heidelberg


Vervliet T.,Catholic University of Leuven | Decrock E.,Ghent University | Molgo J.,French National Center for Scientific Research | Sorrentino V.,University of Siena | And 7 more authors.
Journal of Cell Science | Year: 2014

The anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein not only counteracts apoptosis at the mitochondria by scaffolding proapoptotic Bcl-2-family members, but also acts at the endoplasmic reticulum, thereby controlling intracellular Ca2+ dynamics. Bcl-2 inhibits Ca2+ release by targeting the inositol 1,4,5-trisphosphate receptor (IP3R). Sequence analysis has revealed that the Bcl-2-binding site on the IP3R displays strong similarity with a conserved sequence present in all three ryanodine receptor (RyR) isoforms. We now report that Bcl-2 co-immunoprecipitated with RyRs in ectopic expression systems and in native rat hippocampi, indicating that endogenous RyR-Bcl-2 complexes exist. Purified RyR domains containing the putative Bcl-2-binding site bound fulllength Bcl-2 in pulldown experiments and interacted with the BH4 domain of Bcl-2 in surface plasmon resonance (SPR) experiments, suggesting a direct interaction. Exogenous expression of fulllength Bcl-2 or electroporation loading of the BH4 domain of Bcl-2 dampened RyR-mediated Ca2+ release in HEK293 cell models. Finally, introducing the BH4-domain peptide into hippocampal neurons through a patch pipette decreased RyR-mediated Ca2+ release. In conclusion, this study identifies Bcl-2 as a new inhibitor of RyR-based intracellular Ca2+-release channels. © 2014. Published by The Company of Biologists Ltd. Published by The Company of Biologists Ltd | Journal of Cell Science.


Sestili P.,Urbino University | Ambrogini P.,Urbino University | Barbieri E.,Urbino University | Barbieri E.,Interuniversitary Institute of Myology | And 13 more authors.
Amino Acids | Year: 2016

A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions. © 2015 Springer-Verlag Wien

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