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Aim: Parvalbumin (PV) is a primary calcium buffer in mouse fast skeletal muscle fibers. Previous work showed that PV ablation has a limited impact on cytosolic Ca 2+ ([Ca 2+ ] cyto ) transients and contractile response, while it enhances mitochondrial density and mitochondrial matrix-free calcium concentration ([Ca 2+ ] mito ). Here, we aimed to quantitatively test the hypothesis that mitochondria act to compensate for PV deficiency., Methods: We determined the free Ca 2+ redistribution during a 2 s 60 Hz tetanic stimulation in the sarcoplasmic reticulum, cytosol, and mitochondria. Via a reaction-diffusion Ca 2+ model, we quantitatively evaluated mitochondrial uptake and storage capacity requirements to compensate for PV lack and analyzed possible extracellular export., Results: [Ca 2+ ] mito during tetanic stimulation is greater in knock-out (KO) (1362 ± 392 nM) than in wild-type (WT) (855 ± 392 nM), p < 0.05. Under the assumption of a non-linear intramitochondrial buffering, the model predicts an accumulation of 725 μmoles/L fiber (buffering ratio 1:11 000) in KO, much higher than in WT (137 μmoles/L fiber , ratio 1:4500). The required transport rate via mitochondrial calcium uniporter (MCU) reaches 3 mM/s, compatible with available literature. TEM images of calcium entry units and Mn 2+ quenching showed a greater capacity of store-operated calcium entry in KO compared to WT. However, levels of [Ca 2+ ] cyto during tetanic stimulation were not modulated to variations of extracellular calcium., Conclusions: The model-based analysis of experimentally determined calcium distribution during tetanic stimulation showed that mitochondria can act as a buffer to compensate for the lack of PV. This result contributes to a better understanding of mitochondria's role in modulating [Ca 2+ ] cyto in skeletal muscle fibers., (© 2024 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

RYR1 is the most commonly mutated gene associated with congenital myopathies, a group of early-onset neuromuscular conditions of variable severity. The functional effects of a number of dominant RYR1 mutations have been established; however, for recessive mutations, these effects may depend on multiple factors, such as the formation of a hypomorphic allele, or on whether they are homozygous or compound heterozygous. Here, we functionally characterize a new transgenic mouse model knocked-in for mutations identified in a severely affected child born preterm and presenting limited limb movement. The child carried the homozygous c.14928C>G RYR1 mutation, resulting in the p.F4976L substitution. In vivo and ex vivo assays revealed that homozygous mice fatigued sooner and their muscles generated significantly less force compared with their WT or heterozygous littermates. Electron microscopy, biochemical, and physiological analyses showed that muscles from RyR1 p.F4976L homozygous mice have the following properties: (1) contain fewer calcium release units and show areas of myofibrillar degeneration, (2) contain less RyR1 protein, (3) fibers show smaller electrically evoked calcium transients, and (4) their SR has smaller calcium stores. In addition, single-channel recordings indicate that RyR1 p.F4976L exhibits higher Po in the presence of 100 μM [Ca2+]. Our mouse model partly recapitulates the clinical picture of the homozygous human patient and provides significant insight into the functional impact of this mutation. These results will help understand the pathology of patients with similar RYR1 mutations., (© 2024 Benucci et al.)

Store-operated Ca 2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca 2+ , 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 Ca 2+ . 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.

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity., (© 2022 Michelucci et al.)

Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca 2+ supply to myofibrils during excitation-contraction (EC) coupling (handled by Ca 2+ release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy (CM and EM, respectively) revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged (percentage of severely damaged mitochondria: adults 3.5 ± 1.1%; aged 16.5 ± 3.5%); (b) CRUs that are often misoriented (longitudinal) and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency (adult: 5.1 ± 0.5; aged: 3.9 ± 0.4 n./50 μm 2 ) and size (adult: 362 ± 40 nm; aged: 254 ± 60 nm). The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca 2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.

Proper skeletal muscle function is controlled by intracellular Ca 2+ concentration and by efficient production of energy (ATP), which, in turn, depend on: (a) the release and re-uptake of Ca 2+ from sarcoplasmic-reticulum (SR) during excitation-contraction (EC) coupling, which controls the contraction and relaxation of sarcomeres; (b) the uptake of Ca 2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally (c) the entry of Ca 2+ from the extracellular space via store-operated Ca 2+ entry (SOCE), a mechanism that is important to limit/delay muscle fatigue. Abnormalities in Ca 2+ handling underlie many physio-pathological conditions, including dysfunction in ageing. The specific focus of this review is to discuss the importance of the proper architecture of organelles and membrane systems involved in the mechanisms introduced above for the correct skeletal muscle function. We reviewed the existing literature about EC coupling, mitochondrial Ca 2+ uptake, SOCE and about the structural membranes and organelles deputed to those functions and finally, we summarized the data collected in different, but complementary, projects studying changes caused by denervation and ageing to the structure and positioning of those organelles: a. denervation of muscle fibers-an event that contributes, to some degree, to muscle loss in ageing (known as sarcopenia)-causes misplacement and damage: (i) of membrane structures involved in EC coupling (calcium release units, CRUs) and (ii) of the mitochondrial network; b. sedentary ageing causes partial disarray/damage of CRUs and of calcium entry units (CEUs, structures involved in SOCE) and loss/misplacement of mitochondria; c. functional electrical stimulation (FES) and regular exercise promote the rescue/maintenance of the proper architecture of CRUs, CEUs, and of mitochondria in both denervation and ageing. All these structural changes were accompanied by related functional changes, i.e., loss/decay in function caused by denervation and ageing, and improved function following FES or exercise. These data suggest that the integrity and proper disposition of intracellular organelles deputed to Ca 2+ handling and aerobic generation of ATP is challenged by inactivity (or reduced activity); modifications in the architecture of these intracellular membrane systems may contribute to muscle dysfunction in ageing and sarcopenia.

In the last decades the term Store-operated Ca 2+ entry (SOCE) has been used in the scientific literature to describe an ubiquitous cellular mechanism that allows recovery of calcium (Ca 2+ ) from the extracellular space. SOCE is triggered by a reduction of Ca 2+ content (i.e. depletion) in intracellular stores, i.e. endoplasmic or sarcoplasmic reticulum (ER and SR). In skeletal muscle the mechanism is primarily mediated by a physical interaction between stromal interaction molecule-1 (STIM1), a Ca 2+ sensor located in the SR membrane, and ORAI1, a Ca 2+ -permeable channel of external membranes, located in transverse tubules (TTs), the invaginations of the plasma membrane (PM) deputed to propagation of action potentials. It is generally accepted that in skeletal muscle SOCE is important to limit muscle fatigue during repetitive stimulation. We recently discovered that exercise promotes the assembly of new intracellular junctions that contains colocalized STIM1 and ORAI1, and that the presence of these new junctions increases Ca 2+ entry via ORAI1, while improving fatigue resistance during repetitive stimulation. Based on these findings we named these new junctions Ca 2+ Entry Units (CEUs). CEUs are dynamic organelles that assemble during muscle activity and disassemble during recovery thanks to the plasticity of the SR (containing STIM1) and the elongation/retraction of TTs (bearing ORAI1). Interestingly, similar structures described as SR stacks were previously reported in different mouse models carrying mutations in proteins involved in Ca 2+ handling (calsequestrin-null mice; triadin and junctin null mice, etc.) or associated to microtubules (MAP6 knockout mice). Mutations in Stim1 and Orai1 (and calsequestrin-1) genes have been associated to tubular aggregate myopathy (TAM), a muscular disease characterized by: (a) muscle pain, cramping, or weakness that begins in childhood and worsens over time, and (b) the presence of large accumulations of ordered SR tubes (tubular aggregates, TAs) that do not contain myofibrils, mitochondria, nor TTs. Interestingly, TAs are also present in fast twitch muscle fibers of ageing mice. Several important issues remain un-answered: (a) the molecular mechanisms and signals that trigger the remodeling of membranes and the functional activation of SOCE during exercise are unclear; and (b) how dysfunctional SOCE and/or mutations in Stim1, Orai1 and calsequestrin (Casq1) genes lead to the formation of tubular aggregates (TAs) in aging and disease deserve investigation., (© 2020. The Author(s).)

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of Ca2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca2+ entry, as well as sustained Ca2+ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca2+-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca2+ store content and marked SR Ca2+ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm2 versus 2.0 ± 0.3/100 µm2) and FDB (16.7 ± 1.0/100 µm2 versus 2.7 ± 0.5/100 µm2) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca2+ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca2+ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca2+ transient amplitude, increased dependence of luminal SR Ca2+ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca2+ content by assembling CEUs to promote constitutive and store-operated Ca2+ entry., (© 2020 Michelucci et al.)

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca 2+ entry (SOCE). Here, we report that SOCE, peak Ca 2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca 2+ entry coincident with a modest decrease in total releasable Ca 2+ store content. Importantly, this decrease in releasable Ca 2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca 2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca 2+ dynamics and muscle contractile function during acute exercise., Competing Interests: AM, SB, LP, MG, TT, SM, RD, FP No competing interests declared, (© 2019, Michelucci et al.)

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In fast-twitch fibers from adult mice Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ uptake, optimal force generation, and muscle performance. Taken together our results indicate that muscle activity maintains/improves proper association between CRUs and mitochondria., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Calcium release units (CRUs) and mitochondria control myoplasmic [Ca2+] levels and ATP production in muscle, respectively. We recently reported that these two organelles are structurally connected by tethers, which promote proximity and proper Ca2+ signaling.Here we show that disposition, ultrastructure, and density of CRUs and mitochondria and their reciprocal association are compromised in muscle from aged mice. Specifically, the density of CRUs and mitochondria is decreased in muscle fibers from aged (>24 months) vs. adult (3-12 months), with an increased percentage of mitochondria being damaged and misplaced from their normal triadic position. A significant reduction in tether (13.8 ± 0.4 vs. 5.5 ± 0.3 tethers/100 µm2) and CRU-mitochondrial pair density (37.4 ± 0.8 vs. 27.0 ± 0.7 pairs/100 µm2) was also observed in aged mice. In addition, myoplasmic Ca2+ transient (1.68 ± 0.08 vs 1.37 ± 0.03) and mitochondrial Ca2+ uptake (9.6 ± 0.050 vs 6.58 ± 0.54) during repetitive high frequency tetanic stimulation were significantly decreased. Finally oxidative stress, assessed from levels of 3-nitrotyrosine (3-NT), Cu/Zn superoxide-dismutase (SOD1) and Mn superoxide dismutase (SOD2) expression, were significantly increased in aged mice. The reduced association between CRUs and mitochondria with aging may contribute to impaired cross-talk between the two organelles, possibly resulting in reduced efficiency in activity-dependent ATP production and, thus, to age-dependent decline of skeletal muscle performance.