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

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ 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 [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

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

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

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

Store-operated Ca2+ 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 Ca2+ influx during SR Ca2+ release and leak. SOCE is activated immediately and locally as the [Ca2+ ] of the junctional SR terminal cisternae ([Ca2+ ]jSR ) depletes. [Ca2+ ]jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca2+ ]jSR with RyR1 activity probably depends on the SR Ca2+ -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 Ca2+ in the cytoplasm, preventing net Ca2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca2+ redistribution without changing fibre Ca2+ 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 Ca2+ load, setting a deleterious intracellular environment that compromises the function of the skeletal muscle.

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

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

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

Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ 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 Ca2+ 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 [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ 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 Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function.

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

KEY POINTS: Accumulation of inorganic phosphate (Pi ) 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 Pi into SR are fully established.  We showed that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR .  Pre-treatment of permeabilized myofibres with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi .  The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature. ABSTRACT: Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.

Dantrolene requires Mg2+ to arrest malignant hyperthermia.

Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca2+ transients elicited by MH triggers are unknown. Here, we show that Ca2+ release in the absence of Mg2+ is unaffected by the presence of dantrolene but that dantrolene becomes increasingly effective as cytoplasmic-free [Mg2+] (free [Mg2+]cyto) passes mM levels. Furthermore, we found in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced repetitive Ca2+ waves in the presence of resting levels of free [Mg2+]cyto (1 mM). However, an increase of free [Mg2+]cyto to 1.5 mM could increase the period between Ca2+ waves. These results reconcile previous contradictory reports in muscle fibers and isolated RyRs, where Mg2+ is present or absent, respectively, and define the mechanism of action of dantrolene is to increase the Mg2+ affinity of the RyR (or "stabilize" the resting state of the channel) and suggest that the accumulation of the metabolite Mg2+ from MgATP hydrolysis is required to make dantrolene administration effective in arresting an MH episode.

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

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including ß-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both ß-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

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

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 (BSR). 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 amplitude and propagation rate, in contrast to WT fibers that showed a range of amplitudes 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 BSRand 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.

A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres.

KEY POINTS: Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals. ABSTRACT: The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to < 0.1 mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 µm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the millimolar range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion.

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

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, because of 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.

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

Skeletal muscle fibres are very large and elongated. In response to excitation there must be a rapid and uniform release of Ca(2+) throughout for contraction. To ensure a uniform spread of excitation throughout the fibre to all the Ca(2+) 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.

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

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca(2+)]SR to return to endogenous levels without activation of store overload-induced Ca(2+) release. These results are discussed in the context of how pathophysiological Ca(2+) release such as that occurring in malignant hyperthermia can be generated.

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

Recent findings suggest that Notch-1 signaling contributes to neuronal death in ischemic stroke, but the underlying mechanisms are unknown. Hypoxia inducible factor-1α (HIF-1α), a global regulator of cellular responses to hypoxia, can interact with Notch and modulate its signaling during hypoxic stress. Here we show that Notch signaling interacts with the HIF-1α pathway in the process of ischemic neuronal death. We found that a chemical inhibitor of the Notch-activating enzyme, γ-secretase, and a HIF-1α inhibitor, protect cultured cortical neurons against ischemic stress, and combined inhibition of Notch-1 and HIF-1α further decreased neuronal death. HIF-1α and Notch intracellular domain (NICD) are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and in brain tissue from mice subjected to focal ischemic stroke. Overexpression of NICD and HIF-1α in cultured human neural cells enhanced cell death under ischemia-like conditions, and a HIF-1α inhibitor rescued the cells. RNA interference-mediated depletion of endogenous NICD and HIF-1α also decreased cell death under ischemia-like conditions. Finally, mice treated with inhibitors of γ-secretase and HIF-1α exhibited improved outcome after focal ischemic stroke, with combined treatment being superior to individual treatments. Additional findings suggest that the NICD and HIF-1α collaborate to engage pro-inflammatory and apoptotic signaling pathways in stroke.

Muscle fibre mitochondrial [Ca2+ ] dynamics during Ca2+ waves in RYR1 gain-of-function mouse.

AIM: A fraction of the Ca2+ released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca2+ ] ([Ca2+ ]mito ). This transient [Ca2+ ]mito increase may be important in the resynthesis of ATP and other processes. The resynthesis of ATP in the mitochondria generates heat that can lead to hypermetabolic reactions in muscle with ryanodine receptor 1 (RyR1) variants during the cyclic releasing of SR Ca2+ in the presence of a RyR1 agonist. We aimed to analyse whether the mitochondria of RYR1 variant muscle handles Ca2+ differently from healthy muscle. METHODS: We used confocal microscopy to track mitochondrial and cytoplasmic Ca2+ with fluorescent dyes simultaneously during caffeine-induced Ca2+ waves in extensor digitorum longus muscle fibres from healthy mice and mice heterozygous (HET) for a malignant hyperthermia-causative RYR1 variant. RESULTS: Mitochondrial Ca2+ -transient peaks trailed the peak of cytoplasmic Ca2+ transients by many seconds with [Ca2+ ]mito not increasing by more than 250 nM. A strong linear relationship between cytoplasmic Ca2+ and [Ca2+ ]mito amplitudes was observed in HET RYR1 KI fibres but not wild type (WT). CONCLUSION: Our results indicate that [Ca2+ ]mito change within the nM range during SR Ca2+ release. HET fibre mitochondria are more sensitive to SR Ca2+ release flux than WT. This may indicate post-translation modification differences of the mitochondrial Ca2+ uniporter between the genotypes.

Examination of the subsarcolemmal tubular system of mammalian skeletal muscle fibers.

A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ~500-600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ~95 nm in width with ~60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.

Store-operated Ca²âº entry is not required for store refilling in skeletal muscle.

The present review describes store-operated Ca²âº 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 Ca²âº 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.

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

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 individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ 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.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca(2+) signaling in dystrophic mdx mouse muscle.

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.