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Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

Septins are considered the fourth component of the cytoskeleton with the septin7 isoform playing a critical role in the formation of diffusion barriers in phospholipid bilayers and intra- and extracellular scaffolds. While its importance has already been confirmed in different intracellular processes, very little is known about its role in skeletal muscle. Muscle regeneration was studied in a Sept7 conditional knock-down mouse model to prove the possible role of septin7 in this process. Sterile inflammation in skeletal muscle was induced which was followed by regeneration resulting in the upregulation of septin7 expression. Partial knock-down of Sept7 resulted in an increased number of inflammatory cells and myofibers containing central nuclei. Taken together, our data suggest that partial knock-down of Sept7 hinders the kinetics of muscle regeneration, indicating its crucial role in skeletal muscle functions. [ABSTRACT FROM AUTHOR]

Since the recent discovery of the mechanosensitive Piezo1 channels, many studies have addressed the role of the channel in various physiological or even pathological processes of different organs. Although the number of studies on their effects on the musculoskeletal system is constantly increasing, we are still far from a precise understanding. In this review, the knowledge available so far regarding the musculoskeletal system is summarized, reviewing the results achieved in the field of skeletal muscles, bones, joints and cartilage, tendons and ligaments, as well as intervertebral discs. [ABSTRACT FROM AUTHOR]

In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage‐sensor dihydropyridine receptors. Deficiency in 3‐phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T‐tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1‐deficient mice were compared with the results from a model where propagation of the depolarization along the T‐tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T‐tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium‐induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+‐triggered opening of RyRs provides an alleviating support to the diseased process. Key points: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1.Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease‐associated muscle function failure.Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets.The mathematical modelling approach used in the present work proves that the disease‐associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation–contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/−) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/− mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/− mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described. [ABSTRACT FROM AUTHOR]

In mice a naturally occurring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (MstnCmpt–dl1Abc, Cmpt) labeled by a tremendous increase in body weight along with signs of muscle weakness, easier fatigability, decreased Orai1 expression and store operated calcium entry (SOCE). Here, on the one hand, Cmpt fibers were reconstructed with venus-Orai1 but this failed to restore SOCE. On the other hand, the endogenous Orai1 was silenced in fibers from wild type C57Bl6 mice which resulted in ∼70% of Orai1 being silenced in whole muscle homogenates as confirmed by Western blot, accompanied by an inhibitory effect on the voltage dependence of SR calcium release that manifested in a slight shift toward more positive potential values. This maneuver completely hampered SOCE. Our observations are consistent with the idea that Orai1 channels are present in distinct pools responsible for either a rapid refilling of the SR terminal cisternae connected to each voltage-activated calcium transient, or a slow SOCE associated with an overall depletion of calcium in the SR lumen. Furthermore, when Cmpt cells were loaded with the mitochondrial membrane potential sensitive dye TMRE, fiber segments with depolarized mitochondria were identified covering on average 26.5 ± 1.5% of the fiber area. These defective areas were located around the neuromuscular junction and displayed significantly smaller calcium transients. The ultrastructural analysis of the Cmpt fibers revealed changes in the mitochondrial morphology. In addition, the mitochondrial calcium uptake during repetitive stimulation was higher in the Cmpt fibers. Our results favor the idea that reduced function and/or expression of SOCE partners (in this study Orai1) and mitochondrial defects could play an important role in muscle weakness and degeneration associated with certain pathologies, perhaps including loss of function of the neuromuscular junction and aging. [ABSTRACT FROM AUTHOR]

In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl– content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in μm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in μm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels. [ABSTRACT FROM AUTHOR]

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function. [ABSTRACT FROM AUTHOR]

Key points Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation-contraction coupling (ECC) of mammalian skeletal muscle remains unknown., We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+-sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue., We demonstrate that CB1Rs are not connected to the inositol 1,4,5-trisphosphate pathway either in myotubes or in adult muscle fibres., By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein-mediated way in adult skeletal muscle fibres but not in myotubes., These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs., Abstract Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R-mediated signalling affects the contraction and Ca²⁺ homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+-sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ transients, nor did they alter excitation-contraction coupling. By contrast, in isolated muscle fibres of wild-type mice, although CB1R agonists did not evoke IP3-mediated Ca2+ transients too, they significantly reduced the amplitude of the depolarization-evoked transients in a pertussis-toxin sensitive manner, indicating a Gi/o protein-dependent mechanism. Concurrently, on skeletal muscle fibres isolated from CB1R-knockout animals, depolarization-evoked Ca2+ transients, as well qas Ca2+ release flux via ryanodine receptors (RyRs), and the total amount of released Ca2+ was significantly greater than that from wild-type mice. Our results show that CB1R-mediated signalling exerts both a constitutive and an agonist-mediated inhibition on the Ca2+ transients via RyR, regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase and enhances muscle fatigability, which might decrease exercise performance, thus playing a role in myopathies, and therefore should be considered during the development of new cannabinoid drugs. [ABSTRACT FROM AUTHOR]

Background: As an essential trace element selenium plays a significant role in many physiological functions of the organs. It is found within muscles as selenocystein in selenoprotein N, which is involved in redox-modulated calcium homeostasis and in protection against oxidative stress. Methods: The effects of two different selenium compounds (selenate and NanoSe in 0.5 and 5 ppm concentration for two weeks) on muscle properties of mice were examined by measuring in vivo muscle performance, in vitro force in soleus (SOL) and extensor digitorum longus (EDL) muscles and changes in intracellular Ca2+ concentration in single fibers from flexor digitorum brevis (FDB) muscle.. Western-blot analysis on muscle lysates of EDL and SOL were used to measure the selenoprotein N expression. Control mice received 0.3 ppm Se. Results: While the grip force did not change, 5 ppm selenium diets significantly increased the speed of voluntary running and the daily distance covered. Both forms of selenium increased significantly the amplitude of single twitches in EDL and SOL muscle in a concentration dependent manner. Selenate increased fatigue resistance in SOL. The amplitude of the calcium transients evoked by KCl depolarization increased significantly from the control of 343 ± 44 nM to 671 ± 51 nM in the presence of 0.5 ppm selenate in FDB fibers. In parallel, the rate of calcium release during short depolarizations increased significantly from 28.4 ± 2.2 to 45.5 ± 3.8 and 52.1 ± 1.9 µM/ms in the presence of 0.5 ppm NanoSe and selenate, respectively. In 0.5 ppm concentration both selenium compounds increased significantly the selenoprotein N expression only in EDL muscle. Conclusions: Selenium supplementation augments calcium release from the sarcoplasmic reticulum thus improves skeletal muscle performance. These effects are accompanied by the increased selenoprotein N expression in the muscles which could result in increased oxidative stress tolerance in case of long lasting contraction. [ABSTRACT FROM AUTHOR]

Key points Hypermuscularity associated with naturally occurring mutations in the myostatin gene as found in Compact mice results in increased muscle mass but reduced specific force., The calcium sensitivity of the contractile apparatus as assessed on chemically skinned skeletal muscle fibres under isometric conditions is not altered in these animals., While the resting calcium concentration remains unaffected, depolarization-evoked increases in intracellular calcium concentration are suppressed., Spontaneous calcium release events from sarcoplasmic reticulum are also decreased in frequency, amplitude and spatial spread., Our results suggest that mutations in the myostatin gene are accompanied by alterations in excitation contraction coupling, which manifest as a reduction in sarcoplasmic calcium release., Abstract Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (MstnCmpt-dl1Abc or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca2+ concentration ([Ca2+]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 n m, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 n m, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 Δ F/ F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mMs−1) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 m m s−1) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals. [ABSTRACT FROM AUTHOR]

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise. [ABSTRACT FROM AUTHOR]

Background: Astaxanthin (AX) a marine carotenoid is a powerful natural antioxidant which protects against oxidative stress and improves muscle performance. Retinol and its derivatives were described to affect lipid and energy metabolism. Up to date, the effects of AX and retinol on excitation-contraction coupling (ECC) in skeletal muscle are poorly described. Methods: 18 C57Bl6 mice were divided into two groups: Control and AX supplemented in rodent chow for 4 weeks (AstaReal A1010). In vivo and in vitro force and intracellular calcium homeostasis was studied. In some experiments acute treatment with retinol was employed. Results: The voltage activation of calcium transients (V50) were investigated in single flexor digitorum brevis isolated fibers under patch clamp and no significant changes were found following AX supplementation. Retinol shifted V50 towards more positive values and decreased the peak F/F0 of the calcium transients. The amplitude of tetani in the extensor digitorum longus was significantly higher in AX than in control group. Lastly, the mitochondrial calcium uptake was found to be less prominent in AX. Conclusion: AX supplementation increases in vitro tetanic force without affecting ECC and exerts a protecting effect on the mitochondria. Retinol treatment has an inhibitory effect on ECC in skeletal muscle. [ABSTRACT FROM AUTHOR]

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development. [ABSTRACT FROM AUTHOR]