Antonio Michelucci

Age-dependent uncoupling of mitochondria from Ca 2+ release units in skeletal muscle

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/100μm2) and CRU-mitochondrial pair density (37.4±0.8 vs. 27.0±0.7 pairs/100μm2) 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.

Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice

BackgroundMutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) 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 Ca2+ 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.MethodsWe 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.ResultsAbout 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.ConclusionsOur 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.

Antioxidants Protect Calsequestrin-1 Knockout Mice from Halothane- and Heat-induced Sudden Death

Background:Mice lacking calsequestrin-1 (CASQ1-null), a Ca2+-binding protein that modulates the activity of Ca2+ release in the skeletal muscle, exhibit lethal hypermetabolic episodes that resemble malignant hyperthermia in humans when exposed to halothane or heat stress. Methods:Because oxidative species may play a critical role in malignant hyperthermia crises, we treated CASQ1-null mice with two antioxidants, N-acetylcysteine (NAC, Sigma-Aldrich, Italy; provided ad libitum in drinking water) and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich; administered by intraperitoneal injection), before exposure to halothane (2%, 1 h) or heat (41°C, 1 h). Results:NAC and Trolox significantly protected CASQ1-null mice from lethal episodes, with mortality being 79% (n = 14), 25% (n = 16), and 20% (n = 5) during halothane exposure and 86% (n = 21), 29% (n = 21), and 33% (n = 6) during heat stress in untreated, NAC-treated, and Trolox-treated mice, respectively. During heat challenge, an increase in core temperature in CASQ1-null mice (42.3° ± 0.1°C, n=10) was significantly reduced by both NAC and Trolox (40.6° ± 0.3°C, n = 6 and 40.5° ± 0.2°C, n = 6). NAC treatment of CASQ1-null muscles/mice normalized caffeine sensitivity during in vitro contracture tests, Ca2+ transients in single fibers, and significantly reduced the percentage of fibers undergoing rhabdomyolysis (37.6 ± 2.5%, 38/101 fibers in 3 mice; 11.6 ± 1.1%, 21/186 fibers in 5 mice). The protective effect of antioxidant treatment likely resulted from mitigation of oxidative stress, because NAC reduced mitochondrial superoxide production, superoxide dismutase type-1 expression, and 3-nitrotyrosine expression, and increased both reduced glutathione and reduced glutathione/oxidized glutathione ratio. Conclusion:These studies provide a deeper understanding of the mechanisms that underlie hyperthermic crises in CASQ1-deficient muscle and demonstrate that antioxidant pretreatment may prevent them.

Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia

In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type‐1 (RYR1Y522S/WT) and calsequestrin‐1 knockout (CASQ1‐null), 2 proteins that control Ca2+ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1Y522S/WT and CASQ1‐null mice to an exertional‐stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1Y522S/WT and CASQ1‐null mice, respectively, vs. 0% in wild‐type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1Y522S/WT and CASQ1‐null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca2+dependent activation of calpains and NF‐kB by lowering myoplasmic Ca2+ concentration and nitro‐oxidative stress, parameters that were elevated in RYR1Y522S/WT and CASQ1‐null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.—Michelucci, A., Paolini, C., Boncompagni, S., Canato, M., Reggiani, C., Protasi, F. Strenuous exercise triggers a life‐threatening response in mice susceptible to malignant hyperthermia. FASEB J. 31, 3649–3662 (2017). www.fasebj.org

Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle

Store-operated Ca2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca2+ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca2+ sensor, and Orai1, the Ca2+-permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca2+, compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca2+ with Mg2+ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca2+Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca2+ ions from the extracellular space during repetitive muscle activity.

Estrogens Protect Calsequestrin-1 Knockout Mice from Lethal Hyperthermic Episodes by Reducing Oxidative Stress in Muscle

Oxidative stress has been proposed to play a key role in malignant hyperthermia (MH), a syndrome caused by excessive Ca2+ release in skeletal muscle. Incidence of mortality in male calsequestrin-1 knockout (CASQ1-null) mice during exposure to halothane and heat (a syndrome closely resembling human MH) is far greater than that in females. To investigate the possible role of sex hormones in this still unexplained gender difference, we treated male and female CASQ1-null mice for 1 month, respectively, with Premarin (conjugated estrogens) and leuprolide (GnRH analog) and discovered that during exposure to halothane and heat Premarin reduced the mortality rate in males (79–27% and 86–20%), while leuprolide increased the incidence of mortality in females (18–73% and 24–82%). We then evaluated the (a) responsiveness of isolated muscles to temperature and caffeine, (b) sarcoplasmic reticulum (SR) Ca2+ release in single fibers, and (c) oxidative stress and the expression levels of main enzymes involved in the regulation of the redox balance in muscle. Premarin treatment reduced the temperature and caffeine sensitivity of EDL muscles, normalized SR Ca2+ release, and reduced oxidative stress in males, suggesting that female sex hormones may protect mice from lethal hyperthermic episodes by reducing both the SR Ca2+ leak and oxidative stress.

Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1Y522S/WT Mice

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.

Aerobic Training Prevents Heatstrokes in Calsequestrin-1 Knockout Mice by Reducing Oxidative Stress

Calsequestrin-1 knockout (CASQ1-null) mice suffer lethal episodes when exposed to strenuous exercise and environmental heat, crises known as exertional/environmental heatstroke (EHS). We previously demonstrated that administration of exogenous antioxidants (N-acetylcysteine and trolox) reduces CASQ1-null mortality during exposure to heat. As aerobic training is known to boost endogenous antioxidant protection, we subjected CASQ1-null mice to treadmill running for 2 months at 60% of their maximal speed for 1 h, 5 times/week. When exposed to heat stress protocol (41°C/1 h), the mortality rate of CASQ1-null mice was significantly reduced compared to untrained animals (86% versus 16%). Protection from heatstrokes was accompanied by a reduced increase in core temperature during the stress protocol and by an increased threshold of response to caffeine of isolated extensor digitorum longus muscles during in vitro contracture test. At cellular and molecular levels, aerobic training (i) improved mitochondrial function while reducing their damage and (ii) lowered calpain activity and lipid peroxidation in membranes isolated from sarcoplasmic reticulum and mitochondria. Based on this evidence, we hypothesize that the protective effect of aerobic training is essentially mediated by a reduction in oxidative stress during exposure of CASQ1-null mice to adverse environmental conditions.

Assembly of Calcium Entry Units Improves Muscle Resistance to Fatigue

We discovered that treadmill exercise in-vivo promotes formation in skeletal fibers of new junctions between sarcoplasmic reticulum (SR) and transverse-tubules (TTs) containing STIM1 and Orai1, the two main players in store-operated Ca2+ entry (SOCE). We proposed that these new SR-TT junctions may function as Calcium Entry Units (CEUs) during repetitive muscle activity. Interestingly, in mice lacking Calsequestrin-1 (CASQ1-null) CEUs are constitutively present. In extensor digitorum longus (EDL) of mice, we quantified using electron-microscopy the number of CEUs/100μm2: 2.0±0.3 9.9±0.7; and 39.6±2.1, respectively in control (ctr)WT, exercised (ex)WT (subjected to 1 h of running at increasing speed: from 5 m/min to 25 m/min), and CASQ1-null mice. Higher number of CEUs/area in exWT and CASQ1-null mice correlates with higher expression levels of STIM1 and Orai1 detected by western-blot. We then used a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds) to compare fatigue resistance in EDL muscles in presence or absence of extracellular Ca2+, or in presence of SOCE inhibitors (BTP-2, 2-APB and SKF-96365). Results of these experiments indicated: a) in 2.5 mM Ca2+ external solution, EDL muscles from exWT and CASQ1-null mice exhibited an increased capability to maintain contractile force compared to ctrWT mice (residual force after 15 tetani: 42.9±3.7%, 69.3±3.1% and 128.8±5.4% respectively for ctrWT, exWT, and CASQ1-null EDL muscles); b) in Ca2+-free external solution, muscles from exWT and CASQ1-null mice showed a greater decay of contractile force than ctrWT (residual force after 15 tetani: 30.4±2.4%, 34.7±3.3% and 63.5±2.2% respectively for ctrWT, exWT, and CASQ1-null EDL muscles); c) virtually identical results were obtained in presence of SOCE inhibitors. These data suggest that CEUs provide a preferential pathway for Ca2+ entry during repetitive muscle activity, likely important to limit muscle fatigue.

Role of STIM1 And Orai1 in the Formation of Tubular Aggregates in Ageing Skeletal Muscle Fibers

Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) tubes, form in ageing fast-twitch fibers of mice, preferentially in males. TAs are also found in biopsies from patients affected by TA Myopathy (TAM), a muscle disorder linked to mutations in STIM1 and Orai1 (proteins involved in store-operated Ca2+ entry, SOCE) We have previously shown using Electron Microscopy (EM) that tubes of TAs appear linked by small bridges, resembling aggregated STIM1 molecules. Here, we compared extensor digitorum longus (EDL) muscles from 2 groups of mice (∼4 months, adults and ≥24 months, aged) to determine: a) presence of STIM1 (and Orai1) in TAs; b) the relative contribution of Ca2+ entry to muscle function during repetitive stimulation in ageing muscle. Immunofluorescence indicates that ageing causes STIM1 (a SR protein), but not Orai (that resides in TTs) to accumulate in TAs. This finding is consistent with: a) EM data showing that TTs are rarely seen in TAs; and b) western-bot analysis showing increased expression levels of both STIM1 splicing variants (arbitrary units: STIM1-short=0.87±0.09 vs. 1.18±0.04; STIM1-long=0.81±0.1 vs. 1.11±0.05, respectively adult vs. aged). During a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds): i) in 2.5 mM Ca2+ external solution, EDL muscles from aged mice exhibit a decreased capability to maintain contractile (relative force after 10 tetani in adult vs. aged: 61.0±3.2%, and 49.9±2.4%); ii) in Ca2+-free external solution, aged EDL muscles display a lower decay in contractile force (relative force after 10 tetani in adult vs. aged: 41.0±3.1%, and 53.4±1.5%). Our results indicate that TAs a) accumulate STIM1, which is likely visible as electron-dense strands between tubes; b) while contain STIM1, do not functionally contribute to Ca2+ entry during repetitive stimulation.