Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4-6 month old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 months of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 months) EDL skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared to control mice. Moreover, reduced Q? charge and L-type Ca(2+)current, that are indexes of affected e-c coupling, were observed in diaphragm fibers from 12-15 month old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.
A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.
The loss in muscle mass coupled with a decrease in specific force and shift in fiber composition are hallmarks of aging. Training and regular exercise attenuate the signs of sarcopenia. However, pathologic conditions limit the ability to perform physical exercise. We addressed whether electrical stimulation (ES) is an alternative intervention to improve muscle recovery and defined the molecular mechanism associated with improvement in muscle structure and function. We analyzed, at functional, structural, and molecular level, the effects of ES training on healthy seniors with normal life style, without routine sport activity. ES was able to improve muscle torque and functional performances of seniors and increased the size of fast muscle fibers. At molecular level, ES induced up-regulation of IGF-1 and modulation of MuRF-1, a muscle-specific atrophy-related gene. ES also induced up-regulation of relevant markers of differentiating satellite cells and of extracellular matrix remodeling, which might guarantee shape and mechanical forces of trained skeletal muscle as well as maintenance of satellite cell function, reducing fibrosis. Our data provide evidence that ES is a safe method to counteract muscle decline associated with aging.
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 CASQ2(R33Q/R33Q) (R33Q) mutation.
The histologic features of aging muscle suggest that denervation contributes to atrophy, that immobility accelerates the process, and that routine exercise may protect against loss of motor units and muscle tissue. Here, we compared muscle biopsies from sedentary and physically active seniors and found that seniors with a long history of high-level recreational activity up to the time of muscle biopsy had 1) lower loss of muscle strength versus young men (32% loss in physically active vs 51% loss in sedentary seniors); 2) fewer small angulated (denervated) myofibers; 3) a higher percentage of fiber-type groups (reinnervated muscle fibers) that were almost exclusive of the slow type; and 4) sparse normal-size muscle fibers coexpressing fast and slow myosin heavy chains, which is not compatible with exercise-driven muscle-type transformation. The biopsies from the old physically active seniors varied from sparse fiber-type groupings to almost fully transformed muscle, suggesting that coexpressing fibers appear to fill gaps. Altogether, the data show that long-term physical activity promotes reinnervation of muscle fibers and suggest that decades of high-level exercise allow the body to adapt to age-related denervation by saving otherwise lost muscle fibers through selective recruitment to slow motor units. These effects on size and structure of myofibers may delay functional decline in late aging.
Cardiac calsequestrin (CASQ2) contributes to intracellular Ca(2+) homeostasis by virtue of its low-affinity/high-capacity Ca(2+) binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation-contraction coupling, especially or exclusively upon ?-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2(R33Q/R33Q) and of CASQ2(-/-) mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2(-/-) mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.
Store-operated Ca(2+) entry (SOCE) in skeletal muscle involves signalling between stromal-interacting molecule 1 (STIM1) in the sarcoplasmic reticulum (SR) and Ca(2+) selective Orai1 channels in the sarcolemma. Here we generate transgenic mice with muscle-specific expression of dominant-negative Orai1 (dnOrai1) and demonstrate that Orai1-dependent SOCE promotes growth and limits fatigue in adult skeletal muscle. dnOrai1 mice lack SOCE specifically in muscle but are fertile and thrive well into adulthood. Although muscle ultrastructure, excitation-contraction (EC) coupling, fibre type, and expression of other Ca(2+) regulatory proteins are unaltered, dnOrai1 mice exhibit reduced body weight, muscle mass and fibre cross-sectional area. Importantly, during intense repetitive activity, dnOrai1 mice display increased susceptibility to fatigue at the single fibre, excised muscle and whole-animal levels. We further show that STIM1 and Orai1 proteins co-localize within the triad junction but do not exist in a preassembled context. These results show that Orai1-dependent SOCE has an important physiological role in muscles of adult mice.
The recessive form of catecholaminergic polymorphic ventricular tachycardia is caused by mutations in the cardiac calsequestrin-2 gene; this variant of catecholaminergic polymorphic ventricular tachycardia is less well characterized than the autosomal-dominant form caused by mutations in the ryanodine receptor-2 gene.
The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7(-/-) mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15(-/-) mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.
Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.
Store-operated calcium entry (SOCE) channels play an important role in Ca(2+) signaling. Recently, excessive SOCE was proposed to play a central role in the pathogenesis of malignant hyperthermia (MH), a pharmacogenic disorder of skeletal muscle. We tested this hypothesis by characterizing SOCE current (ISkCRAC) magnitude, voltage dependence, and rate of activation in myotubes derived from two mouse models of anesthetic- and heat-induced sudden death: 1) type 1 ryanodine receptor (RyR1) knock-in mice (Y524S/+) and 2) calsequestrin 1 and 2 double knock-out (dCasq-null) mice. ISkCRAC voltage dependence and magnitude at -80 mV were not significantly different in myotubes derived from wild type (WT), Y524S/+ and dCasq-null mice. However, the rate of ISkCRAC activation upon repetitive depolarization was significantly faster at room temperature in myotubes from Y524S/+ and dCasq-null mice. In addition, the maximum rate of ISkCRAC activation in dCasq-null myotubes was also faster than WT at more physiological temperatures (35-37°C). Azumolene (50 µM), a more water-soluble analog of dantrolene that is used to reverse MH crises, failed to alter ISkCRAC density or rate of activation. Together, these results indicate that while an increased rate of ISkCRAC activation is a common characteristic of myotubes derived from Y524S/+ and dCasq-null mice and that the protective effects of azumolene are not due to a direct inhibition of SOCE channels.
Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca(2+)-binding protein inside SR, calsequestrin. Mitochondrial free Ca(2+)-concentration ([Ca(2+)]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca(2+) release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca(2+)]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca(2+) concentration. The rise in [Ca(2+)]mito during electrical stimulation occurred in 20-30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s(-1). Accordingly, frequency-dependent increase in [Ca(2+)]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca(2+)]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca(2+)]mito transients were increased by inhibition of mitochondrial Na(+)/Ca(2+) exchanger (mNCX). These results provide direct evidence for fast Ca(2+) accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca(2+)-handling and a dependence of mitochondrial Ca(2+)-handling on intracellular (SR) and external Ca(2+) stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca(2+)-leakage.
Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.
Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.
Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca(2+) binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca(2+) during excitation-contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca(2+) release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca(2+) re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca(2+) depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.
Muscle contraction requires ATP and Ca(2+) and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal ("longitudinal mitochondria") to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca(2+) release units (CRUs) or triads ("triadic mitochondria"). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca(2+) in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca(2+) accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 ± 39% after a single tetanus and 412 ± 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca(2+) indicator. Interestingly, sustained mitochondrial Ca(2+) uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca(2+) sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU(-1)·min(-1)·100 ?m(-2)) during postnatal development in direct linear correspondence (r(2) = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca(2+) spark suppression but does not significantly impact mitochondrial Ca(2+) uptake.
Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3 ± 2.0 flashes/1000 ?m²·100 s before and after 5 tetani) and markedly decreased (to 7.7 ± 1.6 flashes/1000 ?m²·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature-dependent increase in mSOF frequency (11.9 ± 0.8 and 19.8 ± 2.6 flashes/1000 ?m²·100 s at 23°C and 37°C) was observed in fibers from RYR1(Y522S/WT) mice, a mouse model of malignant hyperthermia and heat-induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress
The ability to form biofilms contributes significantly to the pathogenesis of many microbial infections, including a variety of ocular diseases often associated with the biofilm formation on foreign materials. Carvacrol (Car.) is an important component of essential oils and recently has attracted much attention pursuant to its ability to promote microbial biofilm disruption. In the present study Car. has been encapsulated in poly(dl-lactide-co-glycolide (PLGA) nanocapsules in order to obtain a suitable drug delivery system that could represent a starting point for developing new therapeutic strategies against biofilm-associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.
We compared structure and function of EDL and Soleus muscles in adult (4-6?m) mice lacking both Calsequestrin (CASQ) isoforms, the main SR Ca²?-binding proteins. Lack of CASQ induced ultrastructural alterations in ~30% of Soleus fibers, but not in EDL. Twitch time parameters were prolonged in both muscles, although tension was not reduced. However, when stimulated for 2?sec at 100?hz, Soleus was able to sustain contraction, while in EDL active tension declined by 70-80%. The results presented in this paper unmask a differential effect of CASQ1&2 ablation in fast versus slow fibers. CASQ is essential in EDL to provide large amount of Ca²? released from the SR during tetanic stimulation. In contrast, Soleus deals much better with lack of CASQ because slow fibers require lower Ca²? amounts and slower cycling to function properly. Nevertheless, Soleus suffers more severe structural damage, possibly because SR Ca²? leak is more pronounced.
Tubular aggregates (TAs), ordered arrays of elongated sarcoplasmic reticulum (SR) tubules, are present in skeletal muscle from patients with myopathies and are also experimentally induced by extreme anoxia. In wild-type mice TAs develop in a clear age-, sex- (male), and fiber type- (fast twitch) dependence. However, the events preceding the appearance of TAs have not been explored. We investigated the sequential stages leading to the initial appearance and maturation of TAs in EDL from male mice. TAs formation requires two temporally distinct steps that operate via different mechanisms. Initially (before 1 year of age), the SR Ca(2+) binding protein calsequestrin (CASQ) accumulates specifically at the I band level causing swelling of free SR cisternae. In the second stage, the enlarged SR sacs at the I band level extend into multiple, longitudinally oriented tubules with a full complement of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCA) in the membrane and CASQ in the lumen. Tubules gradually acquire a regular cylindrical shape and uniform size apparently in concert with partial crystallization of SERCA. Multiple, small TAs associate to form fewer mature TAs of very large size. Interestingly, in fibers from CASQ1-knockout mice abnormal aggregates of SR tubules have different conformation and never develop into ordered aggregates of straight cylinders, possibly due to lack of CASQ accumulation. We conclude that TAs do not arise abruptly but are the final result of a gradually changing SR architecture and we suggest that the crystalline ATPase within the aggregates may be inactive.
Contractile activation in striated muscles requires a Ca(2+) reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca(2+)] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca(2+) signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca(2+) release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca(2+) permeability and inversely to its Ca(2+) buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca(2+) depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca(2+) depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca(2+)], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca(2+), but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca(2+) as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca(2+) storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.
Spinal cord injury causes muscle wasting and loss of function, which are especially severe after complete and permanent damage to lower motor neurons. In a previous cross-sectional study, long-standing denervated muscles were rescued by home-based functional electrical stimulation (h-bFES) training.
Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.
To define the time course and potential effects of electrical stimulation on permanently denervated muscle, we evaluated excitation-contraction coupling (ECC) of rat leg muscles during progression to long-term denervation by ultrastructural analysis, specific binding to dihydropyridine receptors, ryanodine receptor 1 (RYR-1), Ca channels and extrusion Ca pumps, gene transcription and translation of Ca-handling proteins, and in vitro mechanical properties and electrophysiological analyses of sarcolemmal passive properties and L-type Ca current (ICa) parameters. We found that in response to long-term denervation: 1) isolated muscle that is unable to twitch in vitro by electrical stimulation has very small myofibers but may show a slow caffeine contracture; 2) only roughly half of the muscle fibers with "voltage-dependent Ca channel activity" are able to contract; 3) the ECC mechanisms are still present and, in part, functional; 4)ECC-related gene expression is upregulated; and 5) at any time point, there are muscle fibers that are more resistant than others to denervation atrophy and disorganization of the ECC apparatus. These results support the hypothesis that prolonged "resting" [Ca] may drive progression of muscle atrophy to degeneration and that electrical stimulation-induced [Ca] modulation may mimic the lost nerve influence, playing a key role in modifying the gene expression of denervated muscle. Hence, these data provide a potential molecular explanation for the muscle recovery that occurs in response to rehabilitation strategies developed based on empirical clinical observations.
Malignant hyperthermia (MH) and exertional/environmental heat stroke (EHS) in humans present as similar life threatening crises triggered by volatile anaesthetics and strenuous exercise and/or high temperature, respectively. Many families (70-80%) diagnosed with MH susceptibility (MHS), and a few with EHS, are linked to mutations in the gene for the ryanodine receptor type-1 (RyR1), Ca(2+) release channel of the sarcoplasmic reticulum (SR) of skeletal muscle and a key protein in excitation-contraction (EC) coupling. However, mutations in the RyR1 gene are not found in all MH families, suggesting that alternative genes remain to be identified. In our laboratory we have recently characterized a novel knockout model lacking skeletal muscle calsequestrin (CASQ1), a SR Ca(2+)-binding protein that modulates RyR1 function, and investigated whether these mice present a MH/EHS-like phenotype. Ablation of CASQ1 results in remodelling of the EC coupling apparatus and functional changes, which in male mice causes a striking increase in the rate of spontaneous mortality and susceptibility to trigger MH-like lethal episodes in response to halothane and heat stress. The demonstration that ablation of CASQ1 results in MH- and EHS-like lethal episodes validates CASQ1 as a viable candidate gene for linkage analysis in MH and EHS families where mutations in RyR1 are excluded.
Bi-directional calcium (Ca(2+)) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca(2+) stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca(2+) release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca(2+) stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR-mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.
Calsequestrin-1 (CASQ1) is a moderate-affinity, high-capacity Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) terminal cisternae of skeletal muscle. CASQ1 functions as both a Ca(2+)-binding protein and a luminal regulator of ryanodine receptor (RYR1)-mediated Ca(2+) release. Mice lacking skeletal CASQ1 are viable but exhibit reduced levels of releasable Ca(2+) and altered contractile properties. Here we report that CASQ1-null mice exhibit increased spontaneous mortality and susceptibility to heat- and anesthetic-induced sudden death. Exposure of CASQ1-null mice to either 2% halothane or heat stress triggers lethal episodes characterized by whole-body contractures, elevated core temperature, and severe rhabdomyolysis, which are prevented by prior dantrolene administration. The characteristics of these events are remarkably similar to analogous episodes observed in humans with malignant hyperthermia (MH) and animal models of MH and environmental heat stroke (EHS). In vitro studies indicate that CASQ1-null muscle exhibits increased contractile sensitivity to temperature and caffeine, temperature-dependent increases in resting Ca(2+), and an increase in the magnitude of depolarization-induced Ca(2+) release. These results demonstrate that CASQ1 deficiency alters proper control of RYR1 function and suggest CASQ1 as a potential candidate gene for linkage analysis in families with MH/EHS where mutations in the RYR1 gene are excluded.
Inclusion body myositis, the most common muscle disorder in the elderly, is partly characterized by abnormal expression of amyloid precursor protein (APP) and intracellular accumulation of its proteolytic fragments collectively known as ?-amyloid. The present study examined the effects of ?-amyloid accumulation on mitochondrial structure and function of skeletal muscle from transgenic mice (MCK-?APP) engineered to accumulate intramyofiber ?-amyloid. Electron microscopic analysis revealed that a large fraction of myofibers from 2-3-month-old MCK-?APP mice contained numerous, heterogeneous alterations in mitochondria, and other cellular organelles. [(1)H-decoupled](13)C NMR spectroscopy showed a substantial reduction in TCA cycle activity and indicated a switch from aerobic to anaerobic glucose metabolism in the MCK-?APP muscle. Isolated muscle fibers from the MCK-?APP mice also exhibited a reduction in cytoplasmic pH, an increased rate of ROS production, and a partially depolarized plasmalemma. Treatment of MCK-?APP muscle cells with Ru360, a mitochondrial Ca(2+) uniporter antagonist, reversed alterations in the plasmalemmal membrane potential (V(m)) and pH. Consistent with altered redox state of the cells, treatment of MCK-?APP muscle cells with glutathione reversed the effects of ?-amyloid accumulation on Ca(2+) transient amplitudes. We conclude that structural and functional alterations in mitochondria precede the reported appearance of histopathological and clinical features in the MCK-?APP mice and may represent key early events in the pathogenesis of inclusion body myositis.
Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum.
Ca2+ release necessary for muscle contraction occurs at the junctional domain of the sarcoplasmic reticulum (j-SR). It requires the assembly of a large multi-protein complex containing the ryanodine receptor (RyR) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multi-protein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1, TR2 and TR3, that contribute to the localization of triadin at the j-SR. Fluorescence recovery after photobleaching (FRAP) experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Altogether, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multi-protein complex associated with RyR.
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