Store-operated Ca2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca2+, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

The CaV1.1 voltage-gated Ca2+ channel carries L-type Ca2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.

More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference \"2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research\". This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence.

In fast-twitch fibers from adult mice Ca2+ release units (CRUs, i.e. intracellular junctions of excitation-contraction coupling), and mitochondria are structurally linked to each other by small strands, named tethers. We recently showed that aging causes separation of a fraction of mitochondria from CRUs and a consequent impairment of the Ca2+ signaling between the two organelles. However, whether the uncoupling of mitochondria from CRUs is the result of aging per-se or the consequence of reduced muscle activity remains still unclear. Here we studied the association between mitochondria and CRUs: in a) extensor digitorum longus (EDL) muscles from 2 years old mice, either sedentary or trained for 1 year in wheel cages; and b) denervated EDL muscles from adult mice and rats. We analyzed muscle samples using a combination of structural (confocal and electron microscopy), biochemical (assessment of oxidative stress via western blot), and functional (ex-vivo contractile properties, and mitochondrial Ca2+ uptake) experimental procedures. The results collected in structural studies indicate that: a) ageing and denervation result in partial uncoupling between mitochondria and CRUs; b) exercise either maintains (in old mice) or restores (in transiently denervated rats) the association between the two organelles. Functional studies supported the hypothesis that CRU-mitochondria coupling is important for mitochondrial Ca2+ uptake, optimal force generation, and muscle performance. Taken together our results indicate that muscle activity maintains/improves proper association between CRUs and mitochondria.

BACKGROUND: Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca (2+)) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca (2+) buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.
METHODS: We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.
RESULTS: About 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice.
CONCLUSIONS: Our findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.