Bradley Launikonis

Evidence that collaboration between HIF-1α and Notch-1 promotes neuronal cell death in ischemic stroke

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.NBD.2013.10.009

The elusive role of store depletion in the control of intracellular calcium release

Publisher: Springer Science and Business Media LLC

Date: 05-2006

DOI: 10.1007/S10974-006-9082-5

Abstract: The contractile cycle of striated muscles, skeletal and cardiac, is controlled by a cytosolic [Ca2+] transient that requires rapid movements of the ion through channels in the sarcoplasmic reticulum (SR). A functional signature of these channels is their closure after a stereotyped time lapse of Ca2+ release. In cardiac muscle there is abundant evidence that termination of release is mediated by depletion of the Ca2+ store, even if the linkage mechanism remains unknown. By contrast, in skeletal muscle the mechanisms of release termination are not understood. This article reviews measurements of store depletion, the experimental evidence for dependence of Ca2+ release on the [Ca2+] level inside the SR, as well as tests of the molecular nature of putative intra-store Ca2+ sensors. Because Ca2+ sparks exhibit the basic release termination mechanism, much attention is dedicated to the studies of store depletion caused by sparks and its relationship with termination of sparks. The review notes the striking differences in volume, content and buffering power of the stores in cardiac vs. skeletal muscle, differences that explain why functional depletion is much greater for cardiac than skeletal muscle stores. Because in skeletal muscle store depletion is minimal and reduction in store [Ca2+] does not appear to greatly inhibit Ca2+ release, it is concluded that decrease in free SR [Ca2+] does not mediate physiological termination of Ca2+ release in this type of muscle. In spite of the apparent absence of store depletion and its putative channel closing effect, termination of Ca2+ sparks is faster and more robust in skeletal than cardiac muscle. A gating role of a hypothetical "proximate store" constituted by polymers of calsequestrin and associated proteins is invoked in an attempt to preserve a role for store depletion and unify mechanisms in both types of striated muscle.

BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Publisher: Research Square Platform LLC

Date: 24-01-2020

DOI: 10.21203/RS.2.21863/V1

Abstract: Background. BTP2 is known to block Orai1, the Ca 2+ channel of store-operated Ca 2+ entry (SOCE) but no detailed analysis has been undertaken in skeletal muscle, where the drug has been used extensively to study SOCE. Methodology. We trapped a Ca 2+ sensitive dye in the tubular (t-) system of mechanically skinned fibres from rat to define the effect of BTP2 on SOCE in skeletal muscle fibres and used a cytoplasmic rhod-2 to track Ca 2+ release in the presence of BTP2. Results. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of Orai1 channel resting Ca 2+ conductance. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca 2+ ] in the t-system ([Ca 2+ ] t-sys ), shifting from inducing an accumulation of Ca 2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca 2+ ] t-sys . This biphasic effect is not observed in presence of a ryanodine receptor (RyR) inhibitor, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca 2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles independent of extracellular Ca 2+ entry. We determined that the release of Ca 2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Conclusion. Our results show that both Ca 2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.

Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals

Publisher: Proceedings of the National Academy of Sciences

Date: 17-01-2023

DOI: 10.1073/PNAS.2117503120

Abstract: Resting skeletal muscle generates heat for endothermy in mammals but not hibians, while both use the same Ca 2+ -handling proteins and membrane structures to conduct excitation–contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca 2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca 2+ pump, which is lified by increasing RyR1 Ca 2+ leak in mammals, subsequently increasing cytoplasmic [Ca 2+ ] ([Ca 2+ ] cyto ). For thermogenesis to be functional, rising [Ca 2+ ] cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca 2+ leak nor should it compromise the muscle remaining relaxed. To achieve this, Ca 2+ activated, regenerative Ca 2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca 2+ ] cyto and ii) downstream effectors of the SNS increase RyR Ca 2+ leak and subsequently, heat generation. By positioning hibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca 2+ ] cyto under identical conditions optimized for activating regenerative Ca 2+ release as Ca 2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca 2+ ] cyto , resulting in increased SR Ca 2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca 2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca 2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

Upregulation of store-operated Ca²⁺ entry in dystrophic mdx mouse muscle

Publisher: American Physiological Society

Date: 07-2010

DOI: 10.1152/AJPCELL.00524.2009

Abstract: Store-operated Ca 2+ entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca 2+ from the transverse tubule into the junctional cleft during periods of depleting Ca 2+ release. In dystrophic muscle fibers, SOCE may be a source of Ca 2+ overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca 2+ release. To examine this, Ca 2+ in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca 2+ from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca 2+ transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca 2+ concentrations ([Ca 2+ ] SR ). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca 2+ into the fiber for an equivalent depletion of [Ca 2+ ] SR via SOCE, it remains unclear whether this is deleterious.

Confocal imaging of [Ca²⁺] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence

Publisher: Wiley

Date: 30-08-2005

DOI: 10.1113/JPHYSIOL.2005.087973

Ryanodine receptor activity and store‐operated Ca2+ entry: Critical regulators of Ca2+ content and function in skeletal muscle

Publisher: Wiley

Date: 16-03-2022

DOI: 10.1113/JP279512

Abstract: Store‐operated Ca 2+ entry (SOCE) is critical to cell function. In skeletal muscle, SOCE has evolved alongside excitation–contraction coupling (EC coupling) as a result, it displays unique properties compared to SOCE in other cells. The plasma membrane of skeletal muscle is mostly internalized as the tubular system, with the tubules meeting the sarcoplasmic reticulum (SR) terminal cisternae, forming junctions where the proteins that regulate EC coupling and SOCE are positioned. In this review, we describe the properties and roles of SOCE based on direct measurements of Ca 2+ influx during SR Ca 2+ release and leak. SOCE is activated immediately and locally as the [Ca 2+ ] of the junctional SR terminal cisternae ([Ca 2+ ] jSR ) depletes. [Ca 2+ ] jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca 2+ ] jSR with RyR1 activity probably depends on the SR Ca 2+ ‐buffer calsequestrin that is located immediately behind RyR1 inside the SR. This arrangement provides in‐phase activation and deactivation of SOCE with a large dynamic range, allowing precise grading of SOCE flux. The in‐phase activation of SOCE as the SR partially depletes traps Ca 2+ in the cytoplasm, preventing net Ca 2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca 2+ redistribution without changing fibre Ca 2+ content. This condition preserves normal contractile function at the same time as increasing basal metabolic rate. However, higher RyR1 leak drives excess cytoplasmic and mitochondrial Ca 2+ load, setting a deleterious intracellular environment that compromises the function of the skeletal muscle. image

Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise

Publisher: Springer Science and Business Media LLC

Date: 13-02-2017

DOI: 10.1038/NCOMMS14266

Abstract: High-force eccentric exercise results in sustained increases in cytoplasmic Ca 2+ levels ([Ca 2+ ] cyto ), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca 2+ -handling properties. Both t-system components could take up Ca 2+ from the cytoplasm but only transverse tubules supported store-operated Ca 2+ entry. The retention of significant amounts of Ca 2+ within vacuoles provides an effective mechanism to reduce the total content of Ca 2+ within the fibre cytoplasm. We propose this ability can reduce or limit resistance exercise-induced, Ca 2+ -dependent damage to the fibre by the reduction of [Ca 2+ ] cyto to help maintain fibre viability during the period associated with delayed onset muscle soreness.

Concerted vs. sequential. Two activation patterns of vast arrays of intracellular ca²⁺channels in muscle

Publisher: Rockefeller University Press

Date: 26-09-2005

DOI: 10.1085/JGP.200509353

Abstract: To signal cell responses, Ca2+ is released from storage through intracellular Ca2+ channels. Unlike most plasmalemmal channels, these are clustered in quasi-crystalline arrays, which should endow them with unique properties. Two distinct patterns of local activation of Ca2+ release were revealed in images of Ca2+ sparks in permeabilized cells of hibian muscle. In the presence of sulfate, an anion that enters the SR and precipitates Ca2+, sparks became wider than in the conventional, glutamate-based solution. Some of these were “protoplatykurtic” (had a flat top from early on), suggesting an extensive array of channels that activate simultaneously. Under these conditions the rate of production of signal mass was roughly constant during the rise time of the spark and could be as high as 5 μm3 ms−1, consistent with a release current & pA since the beginning of the event. This pattern, called “concerted activation,” was observed also in rat muscle fibers. When sulfate was combined with a reduced cytosolic [Ca2+] (50 nM) these sparks coexisted (and interfered) with a sequential progression of channel opening, probably mediated by Ca2+-induced Ca2+ release (CICR). Sequential propagation, observed only in frogs, may require parajunctional channels, of RyR isoform β, which are absent in the rat. Concerted opening instead appears to be a property of RyR α in the hibian and the homologous isoform 1 in the mammal.

Regulation of Ca2+ sparks by Ca2+ and Mg2+ in mammalian and amphibian muscle. An RyR isoform-specific role in excitation-contraction coupling?

Publisher: Rockefeller University Press

Date: 27-09-2004

DOI: 10.1085/JGP.200409105

Abstract: Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks & μM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4–40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10−7 in the frog or 10−8 in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

Publisher: Wiley

Date: 19-10-2020

DOI: 10.1113/JP279917

Abstract: Accumulation of inorganic phosphate (P i ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P i into SR are fully established. We showed that P i promoted Ca 2+ release at concentrations below 10 m m and decreased it at higher concentrations. This decrease correlated well with that of [Ca 2+ ] SR . Pre‐treatment of permeabilized myofibres with 2 m m Cl − channel blocker 9‐anthracenecarboxylic acid (9AC) inhibited both effects of P i . The biphasic dependence of Ca 2+ release on [P i ] is explained by a direct effect of P i acting on the SR Ca 2+ release channel, combined with the intra‐SR precipitation of Ca 2+ salts. The effects of 9AC demonstrate that P i enters the SR via a Cl − pathway of an as‐yet‐undefined molecular nature. Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P i ). P i diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca 2+ salts, thus contributing to the impairment of Ca 2+ release. Information on the P i entrance pathway is still lacking. In hibian muscles endowed with isoform 3 of the RyR channel, Ca 2+ spark frequency is correlated with the Ca 2+ load of the SR and can be used to monitor this variable. We studied the effects of P i on Ca 2+ sparks in permeabilized fibres of the frog. Relative event frequency ( f / f ref ) rose with increasing [P i ], reaching 2.54 ± 1.6 at 5 m m, and then decreased monotonically, reaching 0.09 ± 0.03 at [P i ] = 80 m m . Measurement of [Ca 2+ ] SR confirmed a decrease correlated with spark frequency at high [P i ]. A large [Ca 2+ ] SR surge was observed upon P i removal. Anion channels are a putative path for P i into the SR. We tested the effect of the chloride channel blocker 9‐anthracenecarboxylic acid (9AC) on P i entrance. 9AC (400 µ m) applied to the cytoplasm produced a non‐significant increase in spark frequency and reduced the P i effects on this parameter. Fibre treatment with 2 m m 9AC in the presence of high cytoplasmic Mg 2+ suppressed the effects of P i on [Ca 2+ ] SR and spark frequency up to 55 m m [P i ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that P i impairs Ca 2+ release by accumulating and precipitating with Ca 2+ inside the SR, thus contributing to myogenic fatigue.

Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

Publisher: American Association for the Advancement of Science (AAAS)

Date: 29-10-2021

DOI: 10.1126/SCIADV.ABI7166

Abstract: RyR1 Ca 2+ leak causes a cascade of events that shifts Ca 2+ to the cytoplasm and mitochondria, supporting force generation.

Store-operated Ca2+ entry is activated by every action potential in skeletal muscle.

Publisher: Springer Science and Business Media LLC

Date: 19-04-2018

DOI: 10.1038/S42003-018-0033-7

Abstract: Store-operated calcium (Ca 2+ ) entry (SOCE) in skeletal muscle is rapidly activated across the tubular system during direct activation of Ca 2+ release. The tubular system is the invagination of the plasma membrane that forms junctions with the sarcoplasmic reticulum (SR) where STIM1, Orai1 and ryanodine receptors are found. The physiological activation of SOCE in muscle is not defined, thus clouding its physiological role. Here we show that the magnitude of a phasic tubular system Ca 2+ influx is dependent on SR Ca 2+ depletion magnitude, and define this as SOCE. Consistent with SOCE, the influx was resistant to nifedipine and BayK8644, and silenced by inhibition of SR Ca 2+ release during excitation. The SOCE transient was shaped by action potential frequency and SR Ca 2+ pump activity. Our results show that SOCE in skeletal muscle acts as an immediate counter-flux to Ca 2+ loss across the tubular system during excitation-contraction coupling.

Effects of β-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

Publisher: Springer Science and Business Media LLC

Date: 22-04-1999

DOI: 10.1007/S004240050867

Abstract: Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents beta-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca2+ under defined conditions. In the presence of 2 microg ml-1 beta-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 microg ml-1, both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 microg ml-1 beta-escin and saponin for 10 min. beta-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca2+ was reduced to a lower level after treatment with beta-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 microg ml-1 beta-escin and 10 microg ml-1 saponin) than toad (10 microg ml-1 beta-escin and 150 microg ml-1 saponin). The reverse order in sensitivities to beta-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of beta-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) beta-escin has a milder action on the surface membrane than saponin (2) beta-escin is a more potent modifier of SR function (3) simple permeabilization of membranes is not sufficient to explain the effects of beta-escin and saponin on muscle membranes and (4) the t-system network within muscle fibres is not a homogeneous compartment.

The accessibility and interconnectivity of the tubular system network in toad skeletal muscle

Publisher: Wiley

Date: 30-10-2008

DOI: 10.1113/JPHYSIOL.2008.155127

Longitudinal and transversal propagation of excitation along the tubular system of rat fast‐twitch muscle fibres studied by high speed confocal microscopy

Publisher: Wiley

Date: 27-01-2012

DOI: 10.1113/JPHYSIOL.2011.221796

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays

Publisher: Springer Science and Business Media LLC

Date: 04-02-2020

DOI: 10.1038/S41598-020-58461-1

Abstract: Elevated cytoplasmic [Ca 2+ ] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca 2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [ 3 H]ryanodine binding to RyR1 at nanomolar Ca 2+ . Both compounds also decrease RyR1 Ca 2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by 50% but only slightly affected Ca 2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance.

Components of activation heat in skeletal muscle

Publisher: Springer Science and Business Media LLC

Date: 25-07-2021

DOI: 10.1007/S10974-019-09547-5

Abstract: Activation heat (q

Store-operated calcium entry remains fully functional in aged mouse skeletal muscle despite a decline in STIM1 protein expression

Publisher: Wiley

Date: 03-05-2011

DOI: 10.1111/J.1474-9726.2011.00706.X

Abstract: Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundant STIM1 and Orai1 in the junctional membranes. The precise role of SOCE in skeletal muscle Ca(2+) homeostasis and excitation-contraction coupling remains to be defined. Regardless, it remains important to determine whether the function and capacity of SOCE changes in aged skeletal muscle. We identified an approximate 40% decline in the expression of the integral SOCE protein, stromal interacting molecule 1 (STIM1), but no such decline in its coupling partner, Orai1, in muscle fibers from aged mice. To determine whether this changed aspects of SOCE functionality in skeletal muscle in aged mice, Ca(2+) in the cytoplasm and t-system were continuously and simultaneously imaged on a confocal microscope during sarcoplasmic reticulum Ca(2+) release and compared to experiments under identical conditions using muscle fibers from young mice. Normal activation, deactivation, Ca(2+) influx, and spatiotemporal characteristics of SOCE were found to persist in skeletal muscle from aged mice. Thus, SOCE remains a robust mechanism in aged skeletal muscle despite the decline in STIM1 protein expression, suggesting STIM1 is in excess in young skeletal muscle.

Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle

Publisher: Proceedings of the National Academy of Sciences

Date: 19-01-2022

DOI: 10.1073/PNAS.2119203119

Abstract: The evolution of mammals to use skeletal muscle as a source of heat allowed them to spread to all parts of the globe. The generation of heat requires increased adenosine triphosphate (ATP) hydrolysis in the resting muscle in a regulated manner, but how this mechanism works is unknown. The results suggest that mammals increase their RyR1 Ca 2+ leak rate to lify a basal ATP turnover rate at the sarcoplasmic reticulum Ca 2+ pump that is higher than that of lower vertebrates. Muscle-based thermogenesis allows regulation of body temperature that is essential for life in mammals and provides a potential pathway for manipulating body weight or temperature by altering metabolic rate.

Growth hormone secretagogues preserve the electrophysiological properties of mouse cardiomyocytes isolated from in vitro ischemia/reperfusion heart.

Publisher: The Endocrine Society

Date: 11-2012

DOI: 10.1210/EN.2012-1404

Abstract: Ischemic heart diseases often induce cardiac arrhythmia with irregular cardiac action potential (AP). This study aims to demonstrate that GH secretagogues (GHS) ghrelin and its synthetic analog hexarelin can preserve the electrophysiological properties of cardiomyocytes experiencing ischemia/reperfusion (I/R). Isolated hearts from adult male mice underwent 20 min global ischemia followed by 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nm) or hexarelin (1 nm) was administered in the perfusion solution either 10 min before or after ischemia, termed pre- or posttreatments. Cardiomyocytes isolated from these hearts were used for whole-cell patch cl ing to measure AP, voltage-gated L-type calcium current (ICaL), transient outward potassium current (Ito), and sodium current (INa). AP litude and duration were significantly decreased by I/R, but GHS treatments maintained their normality. GHS treatments prevented the decrease in ICaL and INa after I/R, thereby maintaining AP litude. Although the significant increase in Ito after I/R partially explained the shortened AP duration, the normalization of it by GHS treatments might contribute to the preservation of AP duration. Phosphorylated p38 and c-Jun NH2-terminal kinase and the downstream active caspase-9 in the cellular apoptosis pathway were significantly increased after I/R but not when GHS treatments were included, whereas phosphorylation of ERK1/2 associated with cell survival showed increase after I/R and a further increase after GHS treatments by binding to its receptor GHS receptor type 1a. These results suggest GHS can not only preserve the electrophysiological properties of cardiomyocytes after I/R but also inhibit cardiomyocyte apoptosis and promote cell survival by modification of MAPK pathways through activating GHS receptor type 1a.

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle

Publisher: Wiley

Date: 10-1997

DOI: 10.1111/J.1469-7793.1997.425BE.X

Abstract: 1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 micrograms ml-1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min-1. Saponin concentrations up to 150 micrograms ml-1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 micrograms ml-1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 micrograms ml-1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min-1 in the presence of 150 micrograms ml-1 saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 microM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 microns) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 micrograms ml-1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat

Publisher: Rockefeller University Press

Date: 11-12-2017

DOI: 10.1085/JGP.201711891

Abstract: The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system’s role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells

Publisher: American Society for Cell Biology (ASCB)

Date: 09-2007

DOI: 10.1091/MBC.E07-01-0024

Abstract: Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29–55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin–dependent mechanism.

A New Selective Pharmacological Enhancer of the Orai1 Ca2+ Channel Reveals Roles for Orai1 in Smooth and Skeletal Muscle Functions

Publisher: American Chemical Society (ACS)

Date: 13-01-2020

DOI: 10.1021/ACSPTSCI.9B00081

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle.

Publisher: Rockefeller University Press

Date: 16-02-2017

DOI: 10.1085/JGP.201611727

Abstract: Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20–30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.

Store‐operated Ca²⁺ entry is not required for store refilling in skeletal muscle

Publisher: Wiley

Date: 25-04-2013

DOI: 10.1111/1440-1681.12078

Abstract: The present review describes store-operated Ca²⁺ entry (SOCE) in skeletal muscle. Fundamental discoveries in the field of skeletal muscle SOCE are described and the techniques that were used to make these. The advantages and limitations in these techniques are discussed to provide a means of questioning and determining the physiological role(s) of SOCE in skeletal muscle. It is concluded that SOCE has little or no role in the filling of the sarcoplasmic reticulum with Ca²⁺ at rest or during a single contracture. It is likely that SOCE is activated during fatigue, although direct measurements of SOCE are lacking and the physiological significance remains uncertain.

BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Publisher: Research Square Platform LLC

Date: 16-04-2020

DOI: 10.21203/RS.3.RS-22948/V1

Abstract: Background . BTP2 is known to block Orai1, the Ca 2+ channel of store-operated Ca 2+ entry (SOCE) but no detailed analysis has been undertaken in skeletal muscle, where the drug has been used extensively to study SOCE. Methodology . We trapped a Ca 2+ sensitive dye in the tubular (t-) system of mechanically skinned fibres from rat to define the effect of BTP2 on SOCE in skeletal muscle fibres and used a cytoplasmic rhod-2 to track Ca 2+ release in the presence of BTP2. Results . In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of a resting Ca 2+ conductance, likely to be through the Orai1 channel. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca 2+ ] in the t-system ([Ca 2+ ] t−sys ), shifting from inducing an accumulation of Ca 2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca 2+ ] t−sys . In absence of functional ryanodine receptors (RyRs), this biphasic effect was not observed, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca 2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles, independently of extracellular Ca 2+ entry impairment. We determined that the release of Ca 2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Finally, we found that BTP2 inhibition of RyR-mediated Ca 2+ release was independent of extracellular or intracellular application of the agent, indicating that BTP2 can impair RyR function in intact muscle. Conclusion . Our results show that both Ca 2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.

Growth hormone secretagogues protect mouse cardiomyocytes from in vitro ischemia/reperfusion injury through regulation of intracellular calcium.

Publisher: Public Library of Science (PLoS)

Date: 06-04-2012

DOI: 10.1371/JOURNAL.PONE.0035265

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Publisher: Springer Science and Business Media LLC

Date: 25-06-2010

DOI: 10.1007/S00424-010-0856-7

Abstract: Store-operated Ca(2+) entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca(2+) from SR. The significant depletion of SR Ca(2+) required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation-contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca(2+) during periods of fibre quiescence. While Ca(2+) in SR remains below the activation threshold for SOCE, a low- litude persistent Ca(2+) influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca(2+) and simple balancing of Ca(2+) within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.

Three-dimensional reconstruction and analysis of the tubular system of vertebrate skeletal muscle

Publisher: The Company of Biologists

Date: 09-2013

DOI: 10.1242/JCS.131565

Abstract: Skeletal muscle fibres are very large and elongated. In response to excitation there must be a rapid and uniform release of Ca2+ throughout for contraction. To ensure a uniform spread of excitation throughout the fibre to all the Ca2+ release sites, the muscle internalizes the plasma membrane, to form the tubular (t-) system. Hence the t-system forms a complex and dense network throughout the fibre that is responsible for excitation–contraction coupling and other signalling mechanisms. However, we currently do not have a very detailed view of this membrane network because of limitations in previously used imaging techniques to visualize it. In this study we serially imaged fluorescent dye trapped in the t-system of fibres from rat and toad muscle using the confocal microscope, and deconvolved and reconstructed these images to produce the first three-dimensional reconstructions of large volumes of the vertebrate t-system. These images showed complex arrangements of tubules that have not been described previously and also allowed the association of the t-system with cellular organelles to be visualized. There was a high density of tubules close to the nuclear envelope because of the close and parallel alignment of the long axes of the myofibrils and the nuclei. Furthermore local fluorescence intensity variations from sub-resolution tubules were converted to tubule diameters. Mean diameters of tubules were 85.9±6.6 and 91.2±8.2 nm, from rat and toad muscle under isotonic conditions, respectively. Under osmotic stress the distribution of tubular diameters shifted significantly in toad muscle only, with change specifically occurring in the transverse but not longitudinal tubules.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.JBC.2021.101412

Corrigendum

Publisher: American Physiological Society

Date: 05-2006

DOI: 10.1152/AJPCELL.00054.2006

Calcium-dependent inactivation terminates calcium release in skeletal muscle of amphibians

Publisher: Rockefeller University Press

Date: 17-03-2008

DOI: 10.1085/JGP.200709870

Abstract: In skeletal muscle of hibians, the cell-wide cytosolic release of calcium that enables contraction in response to an action potential appears to be built of Ca2+ sparks. The mechanism that rapidly terminates this release was investigated by studying the termination of Ca2+ release underlying sparks. In groups of thousands of sparks occurring spontaneously in membrane-permeabilized frog muscle cells a complex relationship was found between litude a and rise time T, which in sparks corresponds to the active time of the underlying Ca2+ release. This relationship included a range of T where a paradoxically decreased with increasing T. Three different methods were used to estimate Ca2+ release flux in groups of sparks of different T. Using every method, it was found that T and flux were inversely correlated, roughly inversely proportional. A simple model in which release sources were inactivated by cytosolic Ca2+ was able to explain the relationship. The predictive value of the model, evaluated by analyzing the variance of spark litude, was found to be high when allowance was made for the out-of-focus error contribution to the total variance. This contribution was estimated using a theory of confocal scanning (Ríos, E., N. Shirokova, W.G. Kirsch, G. Pizarro, M.D. Stern, H. Cheng, and A. González. Biophys. J. 2001. 80:169–183), which was confirmed in the present work by simulated line scanning of simulated sparks. Considering these results and other available evidence it is concluded that Ca2+-dependent inactivation, or CDI, provides the crucial mechanism for termination of sparks and cell-wide Ca2+ release in hibians. Given the similarities in kinetics of release termination observed in cell-averaged records of hibian and mammalian muscle, and in spite of differences in activation mechanisms, CDI is likely to play a central role in mammals as well. Trivially, an inverse proportionality between release flux and duration, in sparks or in global release of skeletal muscle, maintains constancy of the amount of released Ca2+.

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle.

Publisher: Elsevier BV

Date: 07-2019

DOI: 10.1016/J.BBAMCR.2019.02.014

Abstract: Skeletal muscle fibres support store-operated Ca

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy

Publisher: Wiley

Date: 2002

DOI: 10.1113/JPHYSIOL.2001.012920

Abstract: The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09 % (n = 17), 1.41 +/- 0.09 % (n = 12) and 0.83 +/- 0.07 % (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30 % when the tubular system was fully depolarized and decreased by 15 % when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 microm. There was also an increase by 30 % and a decrease by 25 % in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50 % hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9 % of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Junctional membrane Ca ²⁺ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation

Publisher: Proceedings of the National Academy of Sciences

Date: 23-07-2018

DOI: 10.1073/PNAS.1800490115

Abstract: In human muscle with ryanodine receptor (RyR) variants, there is expected to be a change in the basal RyRs Ca 2+ leak that may alter local Ca 2+ dynamics. It is not currently possible to assess these basal Ca 2+ dynamics in human muscle. By trapping a Ca 2+ -sensitive fluorescent dye in the transverse tubular (t)-system of freshly biopsied human skeletal muscle fibers we could detect tiny local RyR-dependent Ca 2+ movements. We show that malignant hyperthermia (MH) causative RyR variant human muscle displays a chronically different RyR Ca 2+ leak which reconfigures the Ca 2+ -handling properties by the t-system. This approach can be used to assess human muscle for function and pathogenicity of other RyR variants of uncertain significance, MH susceptibility, and assessment of drugs targeted to the Ca 2+ -handling proteins.

A quantitative description of tubular system Ca²⁺ handling in fast‐ and slow‐twitch muscle fibres

Publisher: Wiley

Date: 29-02-2016

DOI: 10.1113/JP271658

Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad

Publisher: Wiley

Date: 07-2001

DOI: 10.1111/J.1469-7793.2001.00071.X

Abstract: 1. Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation-contraction (E-C) coupling. The cholesterol content of membranes was manipulated with methyl-beta-cyclodextrin (MbetaCD). 2. In mechanically skinned fibres, depletion of membrane cholesterol with MbetaCD caused a dose- and time-dependent decrease in transverse tubular (t)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mM MbetaCD but were not affected after 2 min in the presence of a 10 mM MbetaCD-1 mM cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MbetaCD : cholesterol ratio at 10 mM MbetaCD, indicating that the inhibitory effect of MbetaCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3-4 h in the presence of 10 mM MbetaCD. 3. The duration of TSDIFRs increased markedly soon (< 2 min) after application of 10 mM MbetaCD and 10 mM MbetaCD-cholesterol complexes, but the Ca(2+) activation properties of the contractile apparatus were minimally affected by 10 mM MbetaCD. The Ca(2+) handling abilities of the sarcoplasmic reticulum appeared to be modified after 10 min exposure to 10 mM MbetaCD. 4. Confocal laser scanning microscopy revealed that the integrity of the t-system was not compromised by either intra- or extracellular application of 10 mM MbetaCD and that a large [Ca(2+)] gradient was maintained across the t-system. 5. Membrane cholesterol depletion caused rapid depolarization of the polarized t-system as shown independently by spontaneous TSDIFRs induced by MbetaCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC(4)(3)) in the presence of MbetaCD. This rapid depolarization of the t-system by cholesterol depletion was not prevented by blocking the Na(+) channels with TTX (10 microM) or the L-type Ca(2+) channels with Co(2+) (5 mM). 6. The results demonstrate that cholesterol is important for maintaining the functional integrity of the t-system and sarcoplasmic reticulum, probably by having specific effects on different membrane proteins that may be directly or indirectly involved in E-C coupling.

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Publisher: Proceedings of the National Academy of Sciences

Date: 24-02-2003

DOI: 10.1073/PNAS.0536227100

Abstract: Examination of store-operated Ca 2+ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP 3 ) receptor acts as a sarcoplasmic reticulum [Ca 2+ ] sensor and mediates SOC by physical coupling without playing a key role in Ca 2+ release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP 3 receptors in skeletal muscle, the role of which with respect to Ca 2+ homeostasis was ill defined until now.

Additional in-series compliance reduces muscle force summation and alters the time course of force relaxation during fixed-end contractions

Publisher: The Company of Biologists

Date: 2016

DOI: 10.1242/JEB.143123

Abstract: There are high mechanical demands placed on skeletal muscles in movements requiring rapid acceleration of the body or its limbs. Tendons are responsible for transmitting muscle forces, but, due to their elasticity, can manipulate the mechanics of the internal contractile apparatus. Shortening of the contractile apparatus against the stretch of tendon affects force generation according to known mechanical properties, however, the extent to which differences in tendon compliance alter force development in response to a burst of electrical impulses is unclear. To establish the influence of series compliance on force summation, we studied electrically evoked doublet contractions in the cane toad peroneus muscle in the presence and absence of a compliant artificial tendon. Additional series compliance reduced tetanic force by two-thirds, a finding predicted based on the force-length property of skeletal muscle. Doublet force and force-time integral expressed relative to the twitch were also reduced by additional series compliance. Active shortening over a larger range of the ascending limb of the force-length curve and at a higher velocity, leading to a progressive reduction in force-generating potential, could be responsible. Muscle-tendon interaction may also explain the accelerated time course of force relaxation in the presence of additional compliance. Our findings suggest that a compliant tendon limits force summation under constant-length conditions. However, high series compliance can be mechanically advantageous when a muscle-tendon unit is actively stretched, permitting muscle fibres to generate force almost isometrically, as shown during stretch-shorten cycles in locomotor activities. Restricting active shortening would likely favour rapid force development.

Ca²⁺sparks and embers of mammalian muscle. Properties of the sources

Publisher: Rockefeller University Press

Date: 30-06-2003

DOI: 10.1085/JGP.200308796

Abstract: Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were “lone embers.” Average spatial width was ∼2 μm in the rat and 1.5 μm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37–160 channels opened in the spark. In simulations, 25–60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 μm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35–260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

Store-operated Ca²⁺entry during intracellular Ca²⁺release in mammalian skeletal muscle

Publisher: Wiley

Date: 08-2007

DOI: 10.1113/JPHYSIOL.2007.135046

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

Publisher: Proceedings of the National Academy of Sciences

Date: 15-09-2017

DOI: 10.1073/PNAS.1707316114

Abstract: Engineered cardiac muscle can be used to promote the structural and functional maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, previous studies have not yet produced cardiac tissues with metabolic and proliferative maturation. Here, we develop a 96-well screening platform and screen for cardiac maturation conditions in engineered cardiac muscle. We found that simulating the postnatal switch in metabolic substrates from carbohydrates to fatty acids promoted a switch in metabolism, DNA damage response, and cell cycle arrest in hPSC-CM. Our study shows that this mechanism can be harnessed to enhance the maturation of human hPSC-CM and cardiac tissues, which has major implications for stem cell sciences, drug discovery, and regenerative medicine.

The Changes in Ca2+ Sparks Associated with Measured Modifications of Intra-store Ca2+ Concentration in Skeletal Muscle

Publisher: Rockefeller University Press

Date: 12-06-2006

DOI: 10.1085/JGP.200609545

Abstract: In cardiac muscle and hibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 μM on average in 10 experiments) and a high value (433 μM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak litude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and litude were statistically significant. The “strength” of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was & %. While the average change in [Ca2+]SR was limited, it reached up to 200% in in idual fibers, without causing massive Ca2+ release or an increase of & .5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.

Activation and propagation of Ca²⁺ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle

Publisher: Wiley

Date: 31-07-2014

DOI: 10.1113/JPHYSIOL.2014.274274

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging

Publisher: The Royal Society

Date: 06-10-2014

DOI: 10.1098/RSIF.2014.0570

Abstract: Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca 2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury

Publisher: Public Library of Science (PLoS)

Date: 07-01-2013

DOI: 10.1371/JOURNAL.PONE.0053528

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

Publisher: Rockefeller University Press

Date: 14-12-2021

DOI: 10.1085/JGP.202012747

Abstract: 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.

A probable role of dihydropyridine receptors in repression of Ca²⁺sparks demonstrated in cultured mammalian muscle

Publisher: American Physiological Society

Date: 02-2006

DOI: 10.1152/AJPCELL.00592.2004

Abstract: To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca 2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca 2+ release. Murine primary cultures were confocally imaged for Ca 2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca 2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice ( mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca 2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 μM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca 2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca 2+ ] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca 2+ ] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca 2+ release channels and/or their susceptibility to Ca 2+ -induced activation, thereby suppressing the production of Ca 2+ sparks.

Examination of the Subsarcolemmal Tubular System of Mammalian Skeletal Muscle Fibers

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.BPJ.2013.04.029

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Publisher: Springer Science and Business Media LLC

Date: 23-02-2023

DOI: 10.1038/S41467-023-36579-W

Abstract: Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis , a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The in idual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca 2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Phasic Store-Operated Ca²⁺ Entry During Excitation-Contraction Coupling in Skeletal Muscle Fibers From Exercised Mice.

Publisher: Frontiers Media SA

Date: 12-11-2020

DOI: 10.3389/FPHYS.2020.597647

A mathematical model to quantify RYR Ca2+ leak and associated heat production in resting human skeletal muscle fibers

Publisher: Rockefeller University Press

Date: 21-03-2022

DOI: 10.1085/JGP.202112994

Abstract: Cycling of Ca2+ between the sarcoplasmic reticulum (SR) and myoplasm is an important component of skeletal muscle resting metabolism. As part of this cycle, Ca2+ leaks from the SR into the myoplasm and is pumped back into the SR using ATP, which leads to the consumption of O2 and generation of heat. Ca2+ may leak through release channels or ryanodine receptors (RYRs). RYR Ca2+ leak can be monitored in a skinned fiber preparation in which leaked Ca2+ is pumped into the t-system and measured with a fluorescent dye. However, accurate quantification faces a number of hurdles. To overcome them, we developed a mathematical model of Ca2+ movement in these preparations. The model incorporated Ca2+ pumps that move Ca2+ from the myoplasm to the SR and from the junctional space (JS) to the t-system, Ca2+ buffering by EGTA in the JS and myoplasm and by buffers in the SR, and Ca2+ leaks from the SR into the JS and myoplasm and from the t-system into the myoplasm. The model accurately simulated Ca2+ uptake into the t-system, the relationship between myoplasmic [Ca2+] and steady-state t-system [Ca2+], and the effect of blocking RYR Ca2+ leak on t-system Ca2+ uptake. The magnitude of the leak through the RYRs would contribute ∼5% of the resting heat production of human muscle. In normal resting fibers, RYR Ca2+ leak makes a small contribution to resting metabolism. RYR-focused pathologies have the potential to increase RYR Ca2+ leak and the RYR leak component of resting metabolism.

Inhibitory Control Over Ca2+ Sparks via Mechanosensitive Channels Is Disrupted in Dystrophin Deficient Muscle but Restored by Mini-Dystrophin Expression

Publisher: Public Library of Science (PLoS)

Date: 04-11-2008

DOI: 10.1371/JOURNAL.PONE.0003644

Ultra-rapid activation and deactivation of store-operated Ca2+ entry in skeletal muscle

Publisher: Elsevier BV

Date: 05-2010

DOI: 10.1016/J.CECA.2010.04.001

Abstract: Skeletal muscle is highly specialized for the rapid delivery of Ca(2+) to the contractile apparatus during excitation-contraction coupling (EC coupling). Previous studies have shown the presence of a relatively fast-activated store-operated Ca(2+) entry (SOCE) mechanism (<1s) to be present in skeletal muscle, unlike the situation occurring in non-excitable cells. We simultaneously imaged [Ca(2+)] in the t-system and cytoplasm in mechanically skinned fibers during SR Ca(2+) release and observed both cell-wide Ca(2+) release and Ca(2+) waves. SOCE activation followed cell-wide Ca(2+) release from high sarcoplasmic reticulum (SR) [Ca(2+)] ([Ca(2+)](SR)) by seconds, consistent with depletion of [Ca(2+)](SR) to an absolute threshold for SOCE and an unformed SOCE complex at high [Ca(2+)](SR). Ca(2+) waves occurred at low [Ca(2+)](SR), close to the threshold for SOCE, minimizing the time between Ca(2+) release and Ca(2+) influx. Local activation of SOCE during Ca(2+) waves occurred in approximately 27ms following local initiation of SR depletion indicating a steep relationship between [Ca(2+)](SR) and SOCE activation. Most of this delay was due to slow release of Ca(2+) from SR, leaving only milliseconds at most for the activation of Ca(2+) entry following store depletion. SOCE was also observed to deactivate effectively instantly during store refilling at low [Ca(2+)](SR). These rapid kinetics of SOCE persisted as subsequent Ca(2+) waves propagated along the fiber. Thus we show for the first time millisecond activation and deactivation of SOCE during low litude [Ca(2+)](SR) oscillations at low [Ca(2+)](SR). To account for the observed Ca(2+) movements we propose the SOCE complex forms during the progressive depletion of [Ca(2+)](SR) prior to reaching the activation threshold of SOCE and this complex remains stable at low [Ca(2+)](SR).

Rapid Ca²⁺ flux through the transverse tubular membrane, activated by individual action potentials in mammalian skeletal muscle

Publisher: Wiley

Date: 14-05-2009

DOI: 10.1113/JPHYSIOL.2009.168682

Leaky ryanodine receptors delay the activation of store overload-induced Ca²⁺ release, a mechanism underlying malignant hyperthermia-like events in dystrophic muscle

Publisher: American Physiological Society

Date: 15-04-2016

DOI: 10.1152/AJPCELL.00366.2015

Abstract: The mouse model of Duchenne muscular dystrophy, the mdx mouse, displays changes in Ca 2+ homeostasis that may lead to the pathology of the muscle. Here we examine the activation of store overload-induced Ca 2+ release (SOICR) in mdx muscle. The activation of SOICR is associated with the depolymerization of the sarcoplasmic reticulum (SR) Ca 2+ buffer calsequestrin and the reduction of SR Ca 2+ buffering power (B SR ). The role of SOICR in healthy and dystrophic muscle is unclear. Using skinned fibers we show that lowering the Mg 2+ concentration can activate discrete Ca 2+ release events that did not necessarily lead to activation of SOICR. However, SOICR waves could propagate into these fiber segments. The average delay to activation of SOICR in mdx fibers was longer than in wild-type (WT) fibers. In the lowered Ca 2+ -buffered environment following large SOICR events, brief waves in mdx fibers displayed a low litude and propagation rate, in contrast to WT fibers that showed a range of litudes correlated with wave propagation rate. The distinct properties of SOICR in mdx fibers were consistent with a ryanodine receptor (RyR) that was leakier to Ca 2+ than in WT. The consequence of delayed SOICR and leaky RyRs is prolonged high B SR and a reduction in free Ca 2+ concentration inside the SR as total SR calcium drops. We present a hypothesis that SOICR activation is required in healthy muscle and that this mechanism works suboptimally in mdx fibers to fail to limit the activation of store-operated Ca 2+ entry.

Tiny changes in cytoplasmic Ca2+ cause large changes in mitochondrial Ca2+: what are the triggers and functional implications?

Publisher: American Physiological Society

Date: 10-2022

DOI: 10.1152/AJPCELL.00092.2022

Abstract: Ca 2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca 2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca 2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca 2+ ] mito and resting [Ca 2+ ] cyto and other mitochondrial Ca 2+ -handling properties of skeletal muscle. An important next step is to understand the triggers for Ca 2+ redistribution between intracellular compartments, which determine the mitochondrial Ca 2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca 2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca 2+ measurements relevant to mitochondrial Ca 2+ -handling, the steady-state relationship between cytoplasmic and mitochondrial Ca 2+ , and the potential implications for Ca 2+ handling by muscle mitochondria and cellular function.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Publisher: Wiley

Date: 10-2002

DOI: 10.1006/CBIR.2002.0942

Abstract: Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-system in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-system open to the outside, the sealed t-system of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres.

Effects of Mg²⁺on Ca²⁺release from sarcoplasmic reticulum of skeletal muscle fibres from yabby (crustacean) and rat

Publisher: Wiley

Date: 07-2000

DOI: 10.1111/J.1469-7793.2000.00299.X

Abstract: 1. The role of myoplasmic [Mg2+] on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in the two major types of crustacean muscle fibres, the tonic, long sarcomere fibres and the phasic, short sarcomere fibres of the fresh water decapod crustacean Cherax destructor (yabby) and in the fast-twitch rat muscle fibres using the mechanically skinned muscle fibre preparation. 2. A robust Ca2+-induced Ca2+-release (CICR) mechanism was present in both long and short sarcomere fibres and 1 mM Mg2+ exerted a strong inhibitory action on the SR Ca2+ release in both fibre types. 3. The SR displayed different properties with respect to Ca2+ loading in the long and the short sarcomere fibres and marked functional differences were identified with respect to Mg2+ inhibition between the two crustacean fibre types. Thus, in long sarcomere fibres, the submaximally loaded SR was able to release Ca2+ when [Mg2+] was lowered from 1 to 0.01 mM in the presence of 8 mM ATPtotal and in the virtual absence of Ca2+ (< 5 nM) even when the CICR was suppressed. In contrast, negligible Ca2+ was released from the submaximally loaded SR of short sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0.01 mM under the same conditions as for the long sarcomere fibres. Nevertheless, the rate of SR Ca2+ release in short sarcomere fibres increased markedly when [Mg2+] was lowered in the presence of [Ca2+] approaching the normal resting levels (50-100 nM). 4. Rat fibres were able to release SR Ca2+ at a faster rate than the long sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0. 01 mM in the virtual absence of Ca2+ but, unlike with yabby fibres, the net rate of Ca2+ release was actually increased for conditions that were considerably less favourable to CICR. 5. In summary, it is concluded that crustacean skeletal muscles have more that one functional type of Ca2+-release channels, that these channels display properties that are intermediate between those of mammalian skeletal and cardiac isoforms, that the inhibition exerted by Mg2+ at rest on the crustacean SR Ca2+-release channels must be removed during excitation-contraction coupling and that, unlike in crustacean fibres, CICR cannot play the major role in the activation of SR Ca2+-release channels in the rat skeletal muscle.

Osmotic Properties of the Sealed Tubular System of Toad and Rat Skeletal Muscle

Publisher: Rockefeller University Press

Date: 23-02-2004

DOI: 10.1085/JGP.200308946

Abstract: A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3–containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190–638 mosmol kg−1, the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality. However, increasing the osmolality above 638 to 2,550 mosmol kg−1 caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg−1, a loss of Ca2+ from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by & %) when the osmolality of the myoplasmic environment changed between 210 and 2,800 mosmol kg−1. Results were also validated with calcein. Clear differences between rat and toad fibers were also found with respect to the t-system permeability for glycerol. Thus, glycerol equilibrated across the rat t-system within seconds to minutes, but was not equilibrated across the t-system of toad fibers even after 20 min. These results have broad implications for understanding osmotic properties of the t-system and reversible vacuolation in muscle fibers. Furthermore, we observed for the first time in mammalian fibers an orderly lateral shift of the t-system networks whereby t-tubule networks to the left of the Z-line crossover to become t-tubule networks to the right of the Z-line in the adjacent sarcomere (and vice versa). This orderly rearrangement can provide a pathway for longitudinal continuity of the t-system along the fiber axis.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca²⁺ signaling in dystrophic mdx mouse muscle

Publisher: American Physiological Society

Date: 09-2012

DOI: 10.1152/AJPCELL.00144.2012

Abstract: The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca 2+ from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca 2+ . The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca 2+ entry (SOCE) can also be activated upon release of SR Ca 2+ . In muscle, the SR Ca 2+ sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca 2+ channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca 2+ influx in mdx compared with WT muscle for similar levels of SR Ca 2+ release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca 2+ via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca 2+ extrusion properties as in the WT muscle.

Depletion “skraps” and dynamic buffering inside the cellular calcium store

Publisher: Proceedings of the National Academy of Sciences

Date: 10-02-2006

DOI: 10.1073/PNAS.0511252103

Abstract: Ca 2+ signals, produced by Ca 2+ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca 2+ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca 2+ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca 2+ release channels have closed. This finding indicates that Ca 2+ is released from a “proximate” compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca 2+ , which was recorded upon stimulation of prolonged, cell-wide Ca 2+ release. An intrastore surge upon induction of Ca 2+ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca 2+ -adsorbing linear polymers of calsequestrin, they could deliver Ca 2+ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca 2+ ] and sustain the concentration gradient that drives release flux.

Dantrolene requires Mg2+ to arrest malignant hyperthermia.

Publisher: Proceedings of the National Academy of Sciences

Date: 03-04-2017

DOI: 10.1073/PNAS.1619835114

Abstract: The nature of overactive Ca 2+ release in malignant hyperthermia (MH) and the mechanism of action of the drug dantrolene that arrests MH events are poorly understood. Here, we show that dantrolene stops overactive Ca 2+ release by increasing the affinity of the ryanodine receptor (RyR) to Mg 2+ . In particular, Ca 2+ waves induced by MH triggers in human muscle are not affected by dantrolene unless Mg 2+ increases above resting levels, a condition met by the increase in MgATP hydrolysis during an MH episode. We suggest that only the combination of dantrolene and increased Mg 2+ can depress overactive Ca 2+ release and the resulting excessive heat production to arrest MH.

Multi-species transcriptomics reveals evolutionary diversity in the mechanisms regulating shrimp tail muscle excitation-contraction coupling

Publisher: Elsevier BV

Date: 08-2020

DOI: 10.1016/J.GENE.2020.144765

University Of Queensland

Location: Australia

View Organisation

La Trobe University

Location: Australia

View Organisation

Calcium Cycling And Heat Generation In Skeletal Muscle Fibres.

Start Date: 2018

End Date: 2021

Funder: Australian Research Council

Calcium Regulation In The Skeletal Muscle Triad And Along The Fibre

Start Date: 2008

End Date: 2009

Funder: Australian Research Council

Muscle Fibre Excitability And Calcium Regulation In Skeletal Muscle Of Amphibians And Mammals

Start Date: 2011

End Date: 2013

Funder: Australian Research Council

Physiological Activation And Targets Of Calcium Signalling In Muscle

Start Date: 2014

End Date: 2018

Funder: Australian Research Council

Membrane Excitability And Cellular Calcium Regulation In The Peripheral Nervous System Under Different (patho)-physiological Conditions And In Inflammatory Disease

Start Date: 2008

End Date: 2008

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180100937

Start Date: 01-2018

End Date: 05-2021

Amount: $530,496.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT140101309

Start Date: 2015

End Date: 12-2018

Amount: $772,104.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220102018

Start Date: 07-2022

End Date: 06-2025

Amount: $608,390.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200100435

Start Date: 06-2020

End Date: 12-2023

Amount: $508,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110102849

Start Date: 2011

End Date: 12-2014

Amount: $340,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0879780

Start Date: 04-2008

End Date: 12-2010

Amount: $165,280.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0882427

Start Date: 2008

End Date: 12-2008

Amount: $131,306.00

Funder: Australian Research Council