Sarcoplasmic Reticulum Depletion Research Articles

Ablation of Calsequestrin-1, Ca2+ unbalance, and susceptibility to heat stroke.

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.

Modulation of Cardiac Alternans by Altered Sarcoplasmic Reticulum Calcium Release: A Simulation Study.

Background: Cardiac alternans is an important precursor to arrhythmia, facilitating formation of conduction block, and re-entry. Diseased hearts were observed to be particularly vulnerable to alternans, mainly in heart failure or after myocardial infarction. Alternans is typically linked to oscillation of calcium cycling, particularly in the sarcoplasmic reticulum (SR). While the role of SR calcium reuptake in alternans is well established, the role of altered calcium release by ryanodine receptors has not yet been studied extensively. At the same time, there is strong evidence that calcium release is abnormal in heart failure and other heart diseases, suggesting that these changes might play a pro-alternans role.Aims: To demonstrate how changes to intracellular calcium release dynamics and magnitude affect alternans vulnerability.Methods: We used the state-of-the-art Heijman–Rudy and O’Hara–Rudy computer models of ventricular myocyte, given their detailed representation of calcium handling and their previous utility in alternans research. We modified the models to obtain precise control over SR release dynamics and magnitude, allowing for the evaluation of these properties in alternans formation and suppression.Results: Shorter time to peak SR release and shorter release duration decrease alternans vulnerability by improved refilling of releasable calcium within junctional SR; conversely, slow release promotes alternans. Modulating the total amount of calcium released, we show that sufficiently increased calcium release may surprisingly prevent alternans via a mechanism linked to the functional depletion of junctional SR during release. We show that this mechanism underlies differences between “eye-type” and “fork-type” alternans, which were observed in human in vivo and in silico. We also provide a detailed explanation of alternans formation in the given computer models, termed “sarcoplasmic reticulum calcium cycling refractoriness.” The mechanism relies on the steep SR load–release relationship, combined with relatively limited rate of junctional SR refilling.Conclusion: Both altered dynamics and magnitude of SR calcium release modulate alternans vulnerability. In particular, slow dynamics of SR release, such as those observed in heart failure, promote alternans. Therefore, acceleration of intracellular calcium release, e.g., via synchronization of calcium sparks, may inhibit alternans in failing hearts and reduce arrhythmia occurrence.

Using Statistical Mechanics to Understand Calcium Sparks

The crucial importance of local Ca signaling for regulation of cardiac muscle contraction was theoretically predicted in 1992 as part of local control theory and validated by the experimental discovery of Ca sparks. Sparks are generated by release channel clusters in the form of localized Ca releases from junctional sarcoplasmic reticulum (SR). As release channels interact, the spark is an emergent behavior of the collective, rather than a property of an individual channel. This nanoscale system of interacting molecules which generates elementary biological signals is particularly interesting because this is where physics meets biology. We have constructed an exact mapping of the release channel cluster to a famous model of an interacting particle system in statistical physics, namely the Ising model. We demonstrated that the channel synchronization that corresponds to termination of signals is described by the same equation as the phase transition associated to the change of magnetic field in an Ising ferromagnet. Our Ising-based theory predicts the critical level of SR [Ca] that is required for spark termination. If the SR depletion does not reach this level the spark does not terminate and becomes metastable. The prediction was confirmed by numerical simulations using 2013 Stern et al. spark model. In an ongoing work, we explore the fine structure of calcium sparks using Peierl's contour analysis to investigate the famous Onsager's phase transition to describe the transition from ordered to disordered sparks as a function of β (effective inverse temperature). The disordered sparks were manifested in numerical simulations by the presence of Ca embers, i.e. small clusters of opened channels. In summary, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release channels), bridging the gap between scales.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

K201 (JTV-519) may prevent abnormal Ca(2+) leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca(2+)-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca(2+) loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca(2+)-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca(2+) and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca(2+) release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca(2+) release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active ("high-activity") RyR1 of SkM reconstituted into bilayers, but it had no effect on "low-activity" channels. Likewise, K201 activated cardiac RyR2 under systolic Ca(2+) conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca(2+) conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca(2+) leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca(2+) conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca(2+) overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca(2+) release.

Spatial and Functional Interactions between SK Channels and L-Type Calcium Channels in Cardiomyocytes

Small conductance Ca2+-activated K+ (SK) channels are gated solely by intracellular Ca2+ and hence, provide a critical link between changes in intracellular Ca2+ and membrane potentials. The identification and functional expression of SK channels in the heart provided additional insights into the regulation of cardiac excitability. Indeed, the crosstalk between Ca2+ signaling and SK channels is critical for shaping cardiac repolarization. Our prior studies documented the physical coupling between SK channels and L-type Ca2+ channels (LTCCs). In addition, functional dependence of SK channel on sarcoplasmic reticulum has also been demonstrated. However, the spatial and functional interactions are less well understood. We hypothesize that LTCCs provide the immediate Ca2+ domain for the activation of cardiac SK channels. Using stimulated emission depletion (STED) microscopy, we gained super-resolution images of SK channels, LTCCs and ryanodine receptors (RyR2) in rabbit ventricular myocytes, revealing the spatial coupling of the three molecules. Moreover, we designed a voltage-clamp protocol to test the functional coupling between SK and LTCCs. EGTA or BAPTA were applied to identify the intracellular Ca2+ domains for SK channel activation. SK currents were activated by Ca2+ influx via LTCCs after depletion of sarcoplasmic reticulum (SR) Ca2+ store. BAPTA removed the functional coupling between SKs and LTCCs. In addition, alterations in SK channel activation kinetics were observed after SR Ca2+ depletion. We conclude that the spatial proximity among SK, LTCCs and RyR2 provides the structural basis for the functional complex formation for SK channel activation. Ca2+ influx through LTCCs provides Ca2+ microdomain for SK channel activation as well as a joint contribution from LTCCs and SR for SK channel activation in cardiomyocytes.Funded by NIH (R01 HL085727 and R01 HL085844 to NC), VA (I01 BX000576 to NC) and AHA (14BGIA18870087 to XZ).

R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats).

Store depletion induces Gαq-mediated PLCβ1 activity to stimulate TRPC1 channels in vascular smooth muscle cells.

Depletion of sarcoplasmic reticulum (SR) Ca2+ stores activates store-operated channels (SOCs) composed of canonical transient receptor potential (TRPC) 1 proteins in vascular smooth muscle cells (VSMCs), which contribute to important cellular functions. We have previously shown that PKC is obligatory for activation of TRPC1 SOCs in VSMCs, and the present study investigates if the classic phosphoinositol signaling pathway involving Gαq-mediated PLC activity is responsible for driving PKC-dependent channel gating. The G-protein inhibitor GDP-β-S, anti-Gαq antibodies, the PLC inhibitor U73122, and the PKC inhibitor GF109203X all inhibited activation of TRPC1 SOCs, and U73122 and GF109203X also reduced store-operated PKC-dependent phosphorylation of TRPC1 proteins. Three distinct SR Ca2+ store-depleting agents, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester, cyclopiazonic acid, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamineed, induced translocations of the fluorescent biosensor GFP-PLCδ1-PH from the cell membrane to the cytosol, which were inhibited by U73122. Knockdown of PLCβ1 with small hairpin RNA reduced both store-operated PLC activity and stimulation of TRPC1 SOCs. Immunoprecipitation studies and proximity ligation assays revealed that store depletion induced interactions between TRPC1 and Gαq, and TRPC1 and PLCβ1. We propose a novel activation mechanism for TRPC1 SOCs in VSMCs, in which store depletion induces formation of TRPC1-Gαq-PLCβ1 complexes that lead to PKC stimulation and channel gating.—Shi, J., Miralles, F., Birnbaumer, L., Large, W. A., Albert, A. P. Store depletion induces Gαq-mediated PLCβ1 activity to stimulate TRPC1 channels in vascular smooth muscle cells.

STIM1–Ca2+ signaling modulates automaticity of the mouse sinoatrial node

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca(2+) release from internal Ca(2+) stores (Ca(2+) clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca(2+) entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca(2+) stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca(2+) current, a reduction in L-type Ca(2+) current, and enhancing the activities of Na(+)/Ca(2+) exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca(2+) dynamics in SANCs that links SR Ca(2+) store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.

The Ryanodine Receptor Patchwork: Knitting Calcium Spark Dynamics

The Ryanodine Receptor Patchwork: Knitting Calcium Spark Dynamics

Functionally Isolated Sarcoplasmic Reticulum Model: Intrinsic Regulation of SR Ca2+ Release and Tetracaine Effect

The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (fractional release, FR) in cardiomyocytes is regulated by the L-type Ca2+ current (ICa,L) and the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR (FISRM), cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Intracellular free Ca2+ concentration ([Ca2+]i) was measured with fluo-3 and Ca2+ transients due to SR release were evoked by 100 ms-long caffeine pulses. FISRM was used to study the intrinsic relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for SR Ca2+ release. After SR depletion by prolonged caffeine application, 1-20 electrical stimuli were applied to attain different [Ca2+]SR (from 55.3±10.0 to 123.0±10.3 μM). The variation in total [Ca2+] (Δ[Ca2+]T) during transients was estimated from [Ca2+]i and Ca2+ buffering parameters. FR (Δ[Ca2+]T/[Ca2+]SR) showed a non-linear relationship with [Ca2+]SR (from 5.4±5.4 to 82.8±7.2%, p< 0.05), as previously observed when SR Ca2+ release was triggered by ICa,L. This behavior was reproduced with a mathematical model of FISRM, after inclusion of intra-SR and cytosolic Ca2+ buffers, which resulted in more realistic simulations. In addition, FISRM was used to study the inhibitory effect of tetracaine on FR. The tetracaine concentration that caused 50% inhibition of FR (IC50) was 58.9±5.7 μM. The FISRM may be a useful experimental model to investigate in intact cells the selective effects on SR Ca2+ release of drugs that also affect sarcolemmal currents, such as tetracaine. Support: CNPq.

Examination of Single Channel RyR Behavior from Long-Lasting Ca2+ Sparks

Calcium sparks are crucial to cardiac excitation-contraction coupling and are due to calcium-induced calcium release via ryanodine receptors (RyRs) located on the sarcoplasmic reticulum (SR). A calcium spark results from the concerted opening of a cluster of RyRs, which generally closes by a robust mechanism as shown by stereotypic spark duration (∼40 ms). However, much longer calcium sparks (up to seconds) can be seen in cells isolated from an animal models of heart failure (1) and when RyRs have been pharmacologically inhibited (2). The mechanism underlying such long-lasting events is unclear. It has been suggested that a lack of local SR calcium depletion (from a combination of decreased release flux and increased SR calcium buffering and/or refilling) is important (2), however it has also been suggested that the activity of RyR sub-clusters (located 1 ms =∼1 um apart) may also sustain release (1). We examined calcium sparks in cells treated with tetracaine to partially inhibit RyRs. Spark-like and long-lasting events occurred at the same location in a line-scan image, suggesting a single release site is able to exhibit both types of behavior. Analysis of long-lasting release events (mean duration = 315 ms) yielded results consistent with a mechanism that involves a reduction in RyR availability and there was no detectable change in the location of the origin of release. These conclusions are supported by a computer model that was able to reproduce long-lasting events when the number of stochastically gating RyRs available was reduced and SR calcium was increased. Large alterations in RyR cluster geometry were not required, but the geometric organization of RyRs in the cluster plays an important role.

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition-a phenomenon we dub "spark metastability." Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Characterization of ryanodine receptor‐gated store‐operated calcium entry in rat pulmonary artery smooth muscle cells

Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs) involves interactions between Ca2+ influx and release pathways. Depletion of sarcoplasmic reticulum (SR) Ca2+ content activates the Ca2+ sensor, stromal interacting molecule (STIM), which induces store‐operated Ca2+ entry (SOCE). Previous studies on SOCE focused on inositol 1,4,5‐trisphosphate receptor (IP3R)‐ related pathways and how ryanodine receptor (RyR)‐gated store regulates SOCE remained largely unknown. Here we characterized the RyR‐gated SOCE in rat PASMCs. Intracellular Ca2+ was measured in the presence of 1 μM nifedipine to prevent voltage‐dependent Ca2+ entry and extracellular Ca2+ was introduced to induce SOCE after 20‐minute store depletion in Ca2+‐free environment. The RyR agonist, 4‐chloro‐m‐cresol (4‐CmC; 1mM), induced SOCE in rat PASMCs. This Ca2+ response was blocked by a low concentration of the SOCE inhibitor, Gd3+ (1 μM). The RyR1 and RyR3 inhibitor, dantrolene (30 μM), did not inhibit 4‐ CmC‐induced SOCE. In addition, maurocalcine (400 nM), a RyR1 agonist, failed to induce RyR‐gated Ca2+ release in rat PASMCs and hence did not activate SOCE. These data shows that RyR2 is the major RyR subtype that mediates SOCE in rat PASMCs. (This project is supported by NIH grant NL‐071835)

Effect of dietary calcium supplementation on store‐operated calcium entry in coronary smooth muscle cells from Ossabaw miniature swine with coronary artery disease

Coronary smooth muscle (CSM) cells of Ossabaw swine in the early stages of coronary artery disease have increased storeoperated calcium/cation entry (SOCE). Pigs (N=24) were fed for 6 months an atherogenic diet containing 0.5% Ca (control, C), or 2% Ca by supplementation with either calcium carbonate (high supplement, HS) or non‐fat milk (high dairy, HD) to determine effects on CSM SOCE. At euthanasia atherosclerosis was quantified with intravascular ultrasound and CSM cells were enzymatically isolated and loaded with fura‐2 to measure cytosolic Ca. The manganese (Mn) quench technique was used to assess SOCE after depletion of the sarcoplasmic reticulum (SR) with 5 mM caffeine. While maintaining SR depletion with 10 μM cyclopiazonic acid, the rate of quenching of fura‐2 fluorescence was measured as the percent change in pixel intensity/time (%Δ/min) to estimate SOCE. Mn quench from 64, 59, and 46 CSM (from 2 pigs/group) was in %Δ/min: C=−0.37±0.24, HD=0.04±0.01, HS=−0.42±0.51. These initial data suggest that Ca supplementation by skim milk prevented the increase in SOCE in CSM in atherosclerosis.(Support: NIH HL062552)

Caveolae and propofol effects on airway smooth muscle

The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca(2+) ([Ca(2+)](i)) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca(2+)](i). We postulated that propofol-induced bronchodilatation involves caveolar disruption. Caveolar fractions of human ASM cells were tested for propofol content. [Ca(2+)](i) responses of ASM cells loaded with fura-2 were performed in the presence of 10 µM histamine with and without clinically relevant concentrations of propofol (10 and 30 μM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca(2+) release were evaluated in zero extracellular Ca(2+) using the blockers Xestospongin C and ryanodine. Store-operated Ca(2+) entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca(2+)](i) and force regulation were also evaluated. Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 µM propofol form decreased [Ca(2+)](i) peak (but not plateau) responses to histamine by ~40%, an effect persistent in zero extracellular Ca(2+). Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca(2+)](i), while propofol blunted [Ca(2+)](i) responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signal-regulated kinase phosphorylation. These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca(2+)](i).

Different Capacity for Store-Operated Ca2+ Entry and Ca2+ Extrusion Across the Plasma Membrane of Wild-Type and Dystrophic mdx Mouse Muscle

Store-operated Ca2+ entry (SOCE) is a ubiquitously expressed signalling system that is highly specialized in skeletal muscle. “Deregulated” SOCE had been proposed as a pathway for Ca2+ entry into dystrophic muscle that leads to fibre degradation. We recently showed that this mechanism remains tightly regulated in mdx mouse muscle but the integral SOCE proteins, STIM1 and Orai1, are upregulated 3-fold (Edwards et al., 2010). We now report that the newly identified isoform STIM1L (Darbellay et al., 2011) is upregulated 1.8-fold in mdx muscle. We found SOCE recorded in skinned fibres was 2-fold greater in mdx compared with WT for the same SR Ca2+ release amplitude. However cytoplasmic fluo-4 transients in depleted intact fibres showed SOCE in the absence of a sarcoplasmic reticulum (SR) Ca2+ pump blocker to be of reduced influx rate in mdx compared to WT fibres. A similar level of SR Ca2+ reloading was detemined in both muscle types following SOCE deactivation. Fura-2 imaging in intact fibres in the presence of 50 μM cyclopiazonic acid (CPA) and no external Ca2+ showed that more Ca2+ remained in the cytoplasm of mdx compared to WT fibres following SR depletion suggesting that Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA) is restricted in mdx. This helps explain reduced SOCE in intact mdx fibres, as the washout of CPA following SR depletion resulted in the greater amount of trapped cytoplasmic Ca2+ re-entering SR to cause a greater degree of SOCE deactivation before externally applied Ca2+ could enter the fibre. These results suggest that store-dependent Ca2+ influx is greater and PMCA is restricted in its capacity to extrude Ca2+ in mdx compared to WT fibres.

Sarcoplasmic Reticulum Calcium Leak in Normal and Dystrophic Skeletal Muscle

Under resting conditions, external calcium is known to enter skeletal muscle cells while calcium stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the resting sarcolemmal calcium entry and in the SR calcium leak are still debated but several lines of evidence suggest that an up-regulation of these calcium fluxes occurs in Duchenne Muscular Dystrophy (DMD). We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the murine model of DMD. Using the cytosolic calcium dye Fura2, we first demonstrated that the rate of calcium increase induced by CPA (cyclopiazonic acid) inhibition of SR Ca2+-ATPases at resting potential is significantly higher in mdx fibers suggesting an elevated SR passive calcium leak. However, in these experiments, sarcolemmal calcium influx may contribute to the CPA-induced calcium increase and another series of experiments indicated that CPA-induced SR calcium leak was deeply modified in the absence of external calcium. Fibers were then loaded with the low affinity calcium dye Fluo5N-AM and dialyzed with 50 mM EGTA to measure intraluminal SR calcium changes. Depolarization pulses evoked voltage-dependent Fluo5N fluorescence decreases followed by a recovery phase which was inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR calcium changes. Voltage-dependence and magnitude of depolarization-induced SR calcium depletion were found to be unchanged in mdx fibers but the rate of the recovery phase that followed depletion was found to be faster, suggesting a higher SR calcium reuptake capacity in mdx fibers. Finally, CPA-induced SR calcium leak at −80 mV was found to be significantly higher in mdx fibers. The elevated SR passive calcium leak may participate to the muscle degenerative process in mdx muscle.

Endothelin-Induced Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells

AbstractAgonist-stimulated waves of elevated cytoplasmic Ca2+ concentration ([Ca2+]i ) regulate blood vessel tone and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were generated by a combination of inositol 1,4,5-trisphosphate (IP3) and Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR); although, some of the mechanistic details remain uncertain. However, these findings were derived indirectly from observing agonist-induced [Ca2+]i fluctuations in the cytoplasm.Here, for the first time, we have recorded Endothelin-1 (ET-1) induced waves of Ca2+ depletion from the SR lumen in vascular smooth muscle cells (VSMCs) using a calsequestrin-targeted Ca2+ indicator. Our findings show that these waves: (1) are due to regenerative CICR by the receptors for IP3 (IP3R), (2) have a marked latency period, (3) are characterized by a transient increase in SR Ca2+ ([Ca2+]SR ) both at the point of origin and at the wave front, (4) proceed with diminishing velocity, and (5) are arrested by the nuclear envelope. Our quantitative model indicates that the gradual decrease in the velocity of the SR depletion wave, in the absence of external Ca2+, results from continuity of the SR luminal network.

Endothelin-Induced Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells

AbstractAgonist-stimulated waves of elevated cytoplasmic Ca2+ concentration ([Ca2+]i ) regulate blood vessel tone and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were generated by a combination of inositol 1,4,5-trisphosphate (IP3) and Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR); although, some of the mechanistic details remain uncertain. However, these findings were derived indirectly from observing agonist-induced [Ca2+]i fluctuations in the cytoplasm.Here, for the first time, we have recorded Endothelin-1 (ET-1) induced waves of Ca2+ depletion from the SR lumen in vascular smooth muscle cells (VSMCs) using a calsequestrin-targeted Ca2+ indicator. Our findings show that these waves: (1) are due to regenerative CICR by the receptors for IP3 (IP3R), (2) have a marked latency period, (3) are characterized by a transient increase in SR Ca2+ ([Ca2+]SR ) both at the point of origin and at the wave front, (4) proceed with diminishing velocity, and (5) are arrested by the nuclear envelope. Our quantitative model indicates that the gradual decrease in the velocity of the SR depletion wave, in the absence of external Ca2+, results from continuity of the SR luminal network.