The Evolution of the Mitochondria-to-Calcium Release Units Relationship in Vertebrate Skeletal Muscles

The spatial relationship between mitochondria and the membrane systems, more specifically the calcium release units (CRUs) of skeletal muscle, is of profound functional significance. CRUs are the sites at which Ca2+ is released from the sarcoplasmic reticulum during muscle activation. Close mitochondrion-CRU proximity allows the organelles to take up Ca2+ and thus stimulate aerobic metabolism. Skeletal muscles of most mammals display an extensive, developmentally regulated, close mitochondrion-CRU association, fostered by tethering links between the organelles. A comparative look at the vertebrate subphylum however shows that this specific association is only present in the higher vertebrates (mammals). Muscles in all other vertebrates, even if capable of fast activity, rely on a less precise and more limited mitochondrion-CRU proximity, despite some tethering connections. This is most evident in fish muscles. Clustering of free subsarcolemmal mitochondria in proximity of capillaries is also more frequently achieved in mammalian than in other vertebrates

Ca2+ Entry Units in a Superfast Fish Muscle

Over the past two decades, mounting evidence has demonstrated that a mechanism known as store-operated Ca2+ entry (SOCE) plays a crucial role in sustaining skeletal muscle contractility by facilitating Ca2+ influx from the extracellular space during sarcoplasmic reticulum (SR) Ca2+ depletion. We recently demonstrated that, in exercised fast-twitch muscle from mice, the incidence of Ca2+ entry units (CEUs), newly described intracellular junctions between dead-end longitudinal transverse tubular (T-tubule) extensions and stacks of sarcoplasmic reticulum (SR) flat cisternae, strictly correlate with both the capability of fibers to maintain contractions during fatigue and enhanced Ca2+ influx via SOCE. Here, we tested the broader relevance of this result across vertebrates by searching for the presence of CEUs in the vocal muscles of a teleost fish adapted for extended, high-frequency activity. Specifically, we examined active vs. inactive superfast sonic muscles of plainfin midshipman (Porichthys notatus). Interestingly, muscles from actively humming territorial males had a much higher incidence of CEU SR stacks relative to territorial males that were not actively vocalizing, strengthening the concept that assembly of these structures is dynamic and use-dependent, as recently described in exercised muscles from mice. Our results support the hypothesis that CEUs represent a conserved mechanism, across vertebrates, for enabling high levels of repetitive muscle activity, and also provide new insights into the adaptive mechanisms underlying the unique properties of superfast midshipman sonic muscles

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes and in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid, beclomethasone dipropionate, increased the phosphorylation (=activation) of extracellular-signal regulated kinase 1&2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5min and depended on the fibre-type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria and involve activation of the MAPK pathway

Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT

Central core disease (CCD) is a congenital myopathy linked to mutations in the ryanodine receptor type 1 (RYR1), the sarcoplasmic reticulum Ca2+ release channel of skeletal muscle. CCD is characterized by formation of amorphous cores within muscle fibers, lacking mitochondrial activity. In skeletal muscle of RYR1Y522S/WT knock-in mice, carrying a human mutation in RYR1 linked to malignant hyperthermia (MH) with cores, oxidative stress is elevated and fibers present severe mitochondrial damage and cores. We treated RYR1Y522S/WT mice with N-acetylcysteine (NAC), an antioxidant provided ad libitum in drinking water for either 2 or 6 months. Our results show that 2 months of NAC treatment starting at 2 months of age, when mitochondrial and fiber damage was still minimal, (i) reduce formation of unstructured and contracture cores, (ii) improve muscle function, and (iii) decrease mitochondrial damage. The beneficial effect of NAC treatment is also evident following 6 months of treatment starting at 4 months of age, when structural damage was at an advanced stage. NAC exerts its protective effect likely by lowering oxidative stress, as supported by the reduction of 3-NT and SOD2 levels. This work suggests that NAC administration is beneficial to prevent mitochondrial damage and formation of cores and improve muscle function in RYR1Y522S/WT mice

Program with last minute abstracts of the Padua Days on Muscle and Mobility Medicine, 27 February – 2 March, 2024 (2024Pdm3)

During the 2023 Padua Days on Muscle and Mobility Medicine the 2024 meeting was scheduled from 28 February to 2 March 2024 (2024Pdm3). During autumn 2023 the program was expanded with Scientific Sessions which will take place over five days (in 2024 this includes February 29), starting from the afternoon of 27 February 2024 in the Conference Rooms of the Hotel Petrarca, Thermae of Euganean Hills (Padua), Italy. As per consolidated tradition, the second day will take place in Padua, for the occasion in the Sala San Luca of the Monastery of Santa Giustina in Prato della Valle, Padua, Italy. Confirming the attractiveness of the Padua Days on Muscle and Mobility Medicine, over 100 titles were accepted until 15 December 2023 (many more than expected), forcing the organization of parallel sessions on both 1 and 2 March 2024. The five days will include lectures and oral presentations of scientists and clinicians from Argentina, Austria, Belgium, Brazil, Bulgaria, Canada, Denmark, Egypt, France, Germany, Iceland, Ireland, Italy, Romania, Russia, Slovenia, Switzerland, UK and USA. Only Australia, China, India and Japan are missing from this edition. But we are confident that authors from those countries who publish articles in the PAGEpress: European Journal of Translational Myology (EJTM: 2022 ESCI Clarivate's Impact Factor: 2.2; SCOPUS Cite Score: 3.2) will decide to join us in the coming years. Together with the program established by 31 January 2024, the abstracts will circulate during the meeting only in the electronic version of the EJTM Issue 34 (1) 2024. See you soon in person at the Hotel Petrarca in Montegrotto Terme, Padua, for the inauguration scheduled the afternoon of 27 February 2024 or on-line for free via Zoom. Send us your email address if you are not traditional participants listed in Pdm3 and EJTM address books

In Vitro Synergism of Colistin and N-acetylcysteine against Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an emerging global opportunistic pathogen, responsible for a wide range of human infections, including respiratory tract infections. Intrinsic multidrug resistance and propensity to form biofilms make S. maltophilia infections recalcitrant to treatment. Colistin is among the second-line options in case of difficult-to-treat S. maltophilia infections, with the advantage of being also administrable by nebulization. We investigated the potential synergism of colistin in combination with N-acetylcysteine (NAC) (a mucolytic agent with antioxidant and anti-inflammatory properties) against S. maltophilia grown in planktonic phase and biofilm. Eighteen S. maltophilia clinical isolates (comprising three isolates from cystic fibrosis (CF) and two trimethoprim-sulfamethoxazole (SXT)-resistant strains) were included. Checkerboard assays showed a synergism of colistin/NAC combinations against the strains with colistin Minimum Inhibitory Concentration (MIC) >2 µg/mL (n = 13), suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Nonetheless, time-kill assays revealed that NAC might potentiate colistin activity also in case of lower colistin MICs. A dose-dependent potentiation of colistin activity by NAC was also clearly observed against S. maltophilia biofilms, also at sub-MIC concentrations. Colistin/NAC combinations, at concentrations likely achievable by topical administration, might represent a valid option for the treatment of S. maltophilia respiratory infections and should be examined further

Local expression of SOD1G93A mutant protein triggers neuromuscular junction dismantlement

The alteration of Reactive Oxygen Species (ROS) homeostasis plays a causal role in several chronic pathology such as aging and neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). Although it is recognized that axon and synapses are first cellular sites of degeneration in ALS disease, controversy exists on whether pathological events initially begin at the NMJs and then, in a dying back phenomena, contribute to motor neuron degeneration. Moreover, the precise molecular mechanisms of pathology-associated deterioration in neuromuscular system have remained elusive (1). Here we provide evidences that muscle specific accumulation of SOD1G93A in the transgenic mice model MLC/SOD1G93A (2) induces mitochondria dysfunction and triggers NMJ dismantlement. Further, we demonstrate that treatment of MLC/SOD1G93A mice with Trolox, a potent antioxidant, is sufficient to rescue mitochondria and NMJ defects in the MLC/SOD1G93A mice, stabilizing muscle-nerve connection. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 revealed the activation of specific Protein Kinase as a downstream player of NMJ dismantlement. Overall our data demonstrate that muscle specific expression of SOD1G93A mutation causes mitochondrial impairment and NMJ dismantlement, suggesting that muscle defects and NMJs alteration precede motor neuron degeneration rather than resulting from it

Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age

Background. Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2R33Q/R33Q (R33Q) mutation. Methods and Results. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Conclusions. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Functional Electrical Stimulation: A Possible Strategy to Improve Muscle Function in Central Core Disease?

Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or cores) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers. Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle. As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups. Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology and electron microscopy. All results point to an improvement in muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function (and possibly reduce muscle loss) in CCD patients