Altering Skeletal Muscle EC Coupling by Ablating the Sarcoplasmic Reticulum Protein JP45 Affects Both Metabolism and Muscle Performance in Old Mice

Muscle contraction requires ATP and Ca2+ 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 Ca2+ 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) Ca2+ in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca2+ 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 Ca2+ indicator. Interestingly, sustained mitochondrial Ca2+ 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 Ca2+ sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU−1·min−1·100 μm−2) during postnatal development in direct linear correspondence ( r2 = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca2+ spark suppression but does not significantly impact mitochondrial Ca2+ uptake.

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Effects of carnosine on contractile apparatus Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in human skeletal muscle fibers

Journal of Applied Physiology ◽

10.1152/japplphysiol.01331.2011 ◽

2012 ◽

Vol 112 (5) ◽

pp. 728-736 ◽

Cited By ~ 66

Author(s):

T. L. Dutka ◽

C. R. Lamboley ◽

M. J. McKenna ◽

R. M. Murphy ◽

G. D. Lamb

Keyword(s):

Skeletal Muscle ◽

Human Muscle ◽

Muscle Performance ◽

Type I ◽

Type Ii ◽

Contractile Apparatus ◽

Muscle Carnosine ◽

Ec Coupling ◽

Type I Fibers ◽

Type Ii Fibers

There is considerable interest in potential ergogenic and therapeutic effects of increasing skeletal muscle carnosine content, although its effects on excitation-contraction (EC) coupling in human muscle have not been defined. Consequently, we sought to characterize what effects carnosine, at levels attained by supplementation, has on human muscle fiber function, using a preparation with all key EC coupling proteins in their in situ positions. Fiber segments, obtained from vastus lateralis muscle of human subjects by needle biopsy, were mechanically skinned, and their Ca2+ release and contractile apparatus properties were characterized. Ca2+ sensitivity of the contractile apparatus was significantly increased by 8 and 16 mM carnosine (increase in pCa50 of 0.073 ± 0.007 and 0.116 ± 0.006 pCa units, respectively, in six type I fibers, and 0.063 ± 0.018 and 0.103 ± 0.013 pCa units, respectively, in five type II fibers). Caffeine-induced force responses were potentiated by 8 mM carnosine in both type I and II fibers, with the potentiation in type II fibers being entirely explicable by the increase in Ca2+ sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibers was beyond that expected from the associated increase in Ca2+ sensitivity of the contractile apparatus and suggestive of increased Ca2+-induced Ca2+ release. Thus increasing muscle carnosine content likely confers benefits to muscle performance in both fiber types by increasing the Ca2+ sensitivity of the contractile apparatus and possibly also by aiding Ca2+ release in type I fibers, helping to lessen or slow the decline in muscle performance during fatiguing stimulation.

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Probenecid affects sarcoplasmic reticulum Ca2+ release and depresses contractile activation in mouse skeletal muscle

The Journal of General Physiology ◽

10.1085/jgp.2021ecc23 ◽

2021 ◽

Vol 154 (9) ◽

Author(s):

Francisco Jaque-Fernandez ◽

Bruno Allard ◽

Laloe Monteiro ◽

Aude Lafoux ◽

Corinne Huchet ◽

...

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Extracellular Space ◽

Purinergic Signaling ◽

Muscle Fibers ◽

Atp Release ◽

Extracellular Medium ◽

Mouse Muscle ◽

Therapeutic Benefits ◽

Ec Coupling

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

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The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle

The Journal of General Physiology ◽

10.1085/jgp.202012747 ◽

2020 ◽

Vol 153 (1) ◽

Author(s):

Aldo Meizoso-Huesca ◽

Bradley S. Launikonis

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Action Potential ◽

Indirect Effects ◽

Ryanodine Receptors ◽

Muscle Cells ◽

Repetitive Stimulation ◽

Ec Coupling ◽

Dependent Inhibition ◽

T System

BTP2 is an inhibitor of the Ca2+ channel Orai1, which mediates store-operated Ca2+ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca2+ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca2+ handling modulators. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of resting extracellular Ca2+ flux. Increasing concentrations of BTP2 caused a shift from inducing accumulation of Ca2+ in the t-system due to Orai1 blocking to reducing the resting [Ca2+] in the sealed t-system. This effect was not observed in the absence of functional ryanodine receptors (RYRs), suggesting that higher concentrations of BTP2 impair RYR function. Additionally, we found that BTP2 impaired action potential–induced Ca2+ release from the sarcoplasmic reticulum during repetitive stimulation without compromising the fiber Ca2+ content. BTP2 was found to have an effect on RYR-mediated Ca2+ release, suggesting that RYR is the point of BTP2-induced inhibition during cycles of EC coupling. The effects of BTP2 on the RYR Ca2+ leak and release were abolished by pre-exposure to saponin, indicating that the effects of BTP2 on the RYR are not direct and require a functional t-system. Our results demonstrate the presence of a SOCE channels–mediated basal Ca2+ influx in healthy muscle fibers and indicate that BTP2 has multiple effects on Ca2+ handling, including indirect effects on the activity of the RYR.

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Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

The Journal of General Physiology ◽

10.1085/jgp.200609730 ◽

2007 ◽

Vol 130 (4) ◽

pp. 379-388 ◽

Cited By ~ 15

Author(s):

Nancy M. Lorenzon ◽

Kurt G. Beam

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Sarcoplasmic Reticulum ◽

Conformational Changes ◽

Ec Coupling ◽

C Terminus ◽

N Terminus ◽

Electrically Evoked Contractions ◽

Evoked Contractions

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca2+ channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR α1S or β1a subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the ∼60-kD streptavidin molecule had access to the β1a N and C termini and to the α1S N terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the α1S C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the β1a N or C terminus, or to the α1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α1S II–III loop abolished such contractions, without affecting agonist-induced Ca2+ release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the α1S II–III loop are necessary for EC coupling in skeletal muscle.

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CARDIAC TROPONIN T MEDIATED AUTOIMMUNE RESPONSE AND ITS ROLE IN SKELETAL MUSCLE AGING

Innovation in Aging ◽

10.1093/geroni/igz038.3231 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S882-S882

Author(s):

Tan Zhang ◽

Xin Feng ◽

Bo Feng ◽

Juan Dong ◽

Karen Haas ◽

...

Keyword(s):

Skeletal Muscle ◽

Cardiac Troponin ◽

Troponin T ◽

Neuromuscular Diseases ◽

Muscle Performance ◽

Autoimmune Response ◽

Cardiac Troponin T ◽

Fast Skeletal Muscle ◽

Age Related ◽

Old Mice

Abstract Cardiac troponin T (cTnT), a key component of contractile machinery essential for muscle contraction, is also expressed in skeletal muscle under certain conditions (e.g. neuromuscular diseases and aging). We have reported that skeletal muscle cTnT regulates neuromuscular junction denervation preferentially in fast skeletal muscle of old mice. Here, we further report that cTnT is also enriched within some myofibers, and/or along microvascular walls in old mice fast skeletal muscle. Strikingly, immunoglobulin G (IgG), together with markers of complement system activation, cell death (necroptosis or apoptosis), and macrophage infiltration, were all found to be co-localized with cTnT and IgG in those areas. In addition, elevated cTnT and IgG are associated with lower dystrophin expression on muscle fiber membrane, lower muscle capillary density, and reduced muscle performance (wire hanging test). Using purified recombinant TnT proteins, we confirmed that only cTnT, but not slow or fast skeletal muscle TnT1 or TnT3, was detected by immunoblot using sera from old (but not young) mice with pre-determined elevated cTnT and IgG in their skeletal muscle, indicating the existence of anti-cTnT autoantibodies in sera (previously found in human blood) and skeletal muscle of old mice. Immunoblotting further revealed that the age related changes in skeletaI muscle cTnT and IgG are more prominent in fast skeletal muscle than in slow. Importantly, elevated cTnT and IgG were also detected in skeletal muscles from 4 older adults (65-70 yrs, IMFIT). Our finding suggests a novel autoimmune mechanism mediated by cTnT that underlies age related skeletal muscle abnormalities and dysfunction.

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Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The Journal of General Physiology ◽

10.1085/jgp.201010523 ◽

2010 ◽

Vol 137 (1) ◽

pp. 43-57 ◽

Cited By ~ 54

Author(s):

Ryan E. Loy ◽

Murat Orynbayev ◽

Le Xu ◽

Zoita Andronache ◽

Simona Apostol ◽

...

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Ryanodine Receptor ◽

Muscle Weakness ◽

Dominant Negative ◽

Release Mechanism ◽

Ion Permeation ◽

Release Channel ◽

Ec Coupling

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

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EXCITATION-CONTRACTION COUPLING AND SARCOPLASMIC RETICULUM FUNCTION IN SKELETAL MUSCLE FIBERS OF OLD MICE

Medicine & Science in Sports & Exercise ◽

10.1097/00005768-200105001-00974 ◽

2001 ◽

Vol 33 (5) ◽

pp. S172

Author(s):

G S. Lynch ◽

D R. Plant

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Muscle Fibers ◽

Excitation Contraction Coupling ◽

Contraction Coupling ◽

Skeletal Muscle Fibers ◽

Old Mice

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Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

The Journal of General Physiology ◽

10.1085/jgp.2021ecc14 ◽

2021 ◽

Vol 154 (9) ◽

Author(s):

Romane Idoux ◽

Christine Berthier ◽

Vincent Jacquemond ◽

Bruno Allard

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Action Potentials ◽

Muscle Fibers ◽

Fast Skeletal Muscle ◽

Fast Kinetics ◽

Ec Coupling ◽

Skeletal Muscle Fibers ◽

Charge Movements ◽

Kinetics Of

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.

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Calcium Ion in Skeletal Muscle: Its Crucial Role for Muscle Function, Plasticity, and Disease

Physiological Reviews ◽

10.1152/physrev.2000.80.3.1215 ◽

2000 ◽

Vol 80 (3) ◽

pp. 1215-1265 ◽

Cited By ~ 544

Author(s):

Martin W. Berchtold ◽

Heinrich Brinkmeier ◽

Markus Müntener

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Binding Proteins ◽

Muscle Fibers ◽

Protein Isoforms ◽

Muscle Performance ◽

High Plasticity ◽

Mammalian Skeletal Muscle ◽

Variable Expression ◽

Functional Features

Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aerobic to fast anaerobic physiology. In addition, skeletal muscle exhibits high plasticity that is based on the potential of the muscle fibers to undergo changes of their cytoarchitecture and composition of specific muscle protein isoforms. Adaptive changes of the muscle fibers occur in response to a variety of stimuli such as, e.g., growth and differentition factors, hormones, nerve signals, or exercise. Additionally, the muscle fibers are arranged in compartments that often function as largely independent muscular subunits. All muscle fibers use Ca2+ as their main regulatory and signaling molecule. Therefore, contractile properties of muscle fibers are dependent on the variable expression of proteins involved in Ca2+ signaling and handling. Molecular diversity of the main proteins in the Ca2+ signaling apparatus (the calcium cycle) largely determines the contraction and relaxation properties of a muscle fiber. The Ca2+ signaling apparatus includes 1) the ryanodine receptor that is the sarcoplasmic reticulum Ca2+ release channel, 2) the troponin protein complex that mediates the Ca2+ effect to the myofibrillar structures leading to contraction, 3) the Ca2+pump responsible for Ca2+ reuptake into the sarcoplasmic reticulum, and 4) calsequestrin, the Ca2+storage protein in the sarcoplasmic reticulum. In addition, a multitude of Ca2+-binding proteins is present in muscle tissue including parvalbumin, calmodulin, S100 proteins, annexins, sorcin, myosin light chains, β-actinin, calcineurin, and calpain. These Ca2+-binding proteins may either exert an important role in Ca2+-triggered muscle contraction under certain conditions or modulate other muscle activities such as protein metabolism, differentiation, and growth. Recently, several Ca2+signaling and handling molecules have been shown to be altered in muscle diseases. Functional alterations of Ca2+ handling seem to be responsible for the pathophysiological conditions seen in dystrophinopathies, Brody's disease, and malignant hyperthermia. These also underline the importance of the affected molecules for correct muscle performance.

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