A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

A model of propagating calcium-induced calcium release mediated by calcium diffusion

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading.     

Fura-2 antagonises calcium-induced calcium release

Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.     

Model of calcium-induced calcium release mechanism in cardiac cells

A model with which to elucidate the mechanism of Ca²⁺ release from, and Ca²⁺ loading in the sarcoplasmic reticulum (SR) by Ca²⁺ current (I _Ca) in cardiac cells is proposed. The SR is assumed to be comprised of three functional subcompartments: (1) the main calcium store (MCS), which contains most of the calcium (both free and bound); (2) the releasable terminal (RT), which contains the calcium readily available for release; and (3) the longitudinal network of the SR (LSR), which sequesters and the transfers the sarcoplasmic calcium to the RT. A rapid increase of the Ca²⁺ concentration at the outer surface of the SR (Ca_e) due to the fast component ofI _Ca activates and inactivates this surface, inducing the release of Ca²⁺ from the RT to the sarcoplasmic space. The RT in turn is further activated and inactivated by a increase in the concentration of sarcoplasmic Ca²⁺. The Ca²⁺ in the sarcoplasmic space is then sequestered by the LSR, leading to the reactivation of the RT. Further increase of Ca_e due to the slow component ofI _Ca enhances the entry of Ca²⁺ into the MCS to be bound by the binding substance. The free Ca²⁺ released from the Ca-binding substance complex is transferred to the RT for subsequent release. The activation, inactivation and reactivation are Ca²⁺-mediated and time-dependent. The proposed model yields simulation of the many events qualitatively similar to those observed experimentally in skinned cardiac cells.     

Calcium wave propagation by calcium-induced calcium release: An unusual excitable system

We discuss in detail the behaviour of a model, proposed by Goldbeteret al. (1990.Proc. natn. Acad. Sci. 87, 1461–1465), for intracellular calcium wave propagation by calcium-induced calcium release, focusing our attention on excitability and the propagation of waves in one spatial dimension. The model with no diffusion behaves like a generic excitable system, and threshold behaviour, excitability and oscillations can be understood within this general framework. However, when diffusion is included, the model no longer behaves like a generic excitable system; the fast and slow variables are not distinct and previous results on excitable systems do not necessarily apply. We consider a piecewise linear simplification of the model, and construct travelling pulse and periodic plane wave solutions to the simplified model. The analogous behaviour in the full model is studied numerically. Goldbeter's model for calciuminduced calcium release is an excitable system of a type not previously studied in detail.     

Teaching calcium-induced calcium release in cardiomyocytes using a classic paper by Fabiato

This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In closing, technical issues associated with the skinned cell model are mentioned. Based on this review article, teaching and learning points are put forth in this article to highlight two concepts: 1) the regulatory mechanisms of CICR in cardiomyocytes and 2) the recognition of contradicting hypotheses and limitations in experimental design. The first concept is certainly an important one for physiology students. The second concept is universally applicable to researchers in all fields of science. It is thus the aim of this article to cultivate a rewarding teaching and learning experience for both instructors and students.     

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.     

The voltage-sensitive release mechanism of excitation contraction coupling in rabbit cardiac muscle is explained by calcium-induced calcium release

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37 degrees C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from -40 to -60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by -10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and CdCl2 and blocked completely by NiCl2. "Tail contractions" persisted in the presence of nifedipine and CdCl2 but were blocked completely by NiCl2. Block of early outward current by 4-aminopyridine and 4-acetoamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CdCl2. Inward current did not persist in the presence of NiCl2. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [Na+] and to the outward Na+/Ca2+ exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of Na+/Ca2+ exchange-mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 microM) and by enhancing sarcoplasmic reticulum Ca2+ content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the Ca2+ "trigger" supplied by unblocked L-type Ca2+ channels and Na+/Ca2+ exchange.     

Quinacrine inhibits the calcium-induced calcium release in heavy sarcoplasmic reticulum vesicles

Quinacrine is a fluorescence probe useful for studying the effect of local anesthetics. The interaction of quinacrine and sarcoplasmic reticulum membranes measured by fluorescence spectroscopy indicates the presence of a saturable binding site. Typical local anesthetics are able to displace quinacrine bound to heavy sarcoplasmic reticulum membranes. The effectiveness of that displacement decreases in the order dibucaine greater than tetracaine greater than benzocaine greater than lidocaine greater than procaine greater than procainamide, indicating that the size and hydrophobicity of quinacrine are major determinants in the binding process. The use of radioactive tracer and a rapid filtration technique reveals that quinacrine interacts, at lower concentrations, with sarcoplasmic reticulum membranes by blocking the Ca2+-induced Ca2+ release. Higher quinacrine concentrations also affect the Ca2+-pump activity.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

A simplified local control model of calcium-induced calcium release in cardiac ventricular myocytes

Calcium (Ca2+)-induced Ca2+ release (CICR) in cardiac myocytes exhibits high gain and is graded. These properties result from local control of Ca2+ release. Existing local control models of Ca2+ release in which interactions between L-Type Ca2+ channels (LCCs) and ryanodine-sensitive Ca2+ release channels (RyRs) are simulated stochastically are able to reconstruct these properties, but only at high computational cost. Here we present a general analytical approach for deriving simplified models of local control of CICR, consisting of low-dimensional systems of coupled ordinary differential equations, from these more complex local control models in which LCC-RyR interactions are simulated stochastically. The resulting model, referred to as the coupled LCC-RyR gating model, successfully reproduces a range of experimental data, including L-Type Ca2+ current in response to voltage-clamp stimuli, inactivation of LCC current with and without Ca2+ release from the sarcoplasmic reticulum, voltage-dependence of excitation-contraction coupling gain, graded release, and the force-frequency relationship. The model does so with low computational cost.     

Tau inhibits mitochondrial calcium efflux and makes neurons vulnerable to calcium-induced cell death

Aggregation or phosphorylation of the microtubule-associated protein tau is the pathological hallmark in a number of diseases termed tauopathies, which include the most common neurodegenerative disorder, Alzheimer’s disease; or frontotemporal dementia, linked to mutations in the gene MAPT encoding tau. Although misfolded tau has strong familial and histopathological (as in intracellular tangles) association with neurodegenerative disorders, the cellular mechanism of tau-induced pathology remains to be controversial. Here we studied the effect of tau on the cytosolic and mitochondrial calcium homeostasis using primary cortical cultures treated with the protein and iPSC-derived neurons bearing the 10 + 16 MAPT mutation linked to frontotemporal dementia. We found that incubation of the primary cortical co-cultures of neurons and astrocytes with tau induced spontaneous Ca²⁺ oscillations in the neurons, which were also observed in iPSC-neurons with the 10 + 16 MAPT mutation. Importantly, tau inhibited mitochondrial calcium efflux via the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) in both neurons and astrocytes. This inhibition led to mitochondrial depolarisation in response to physiological and pathological calcium stimuli and made these cells vulnerable to calcium-induced caspase 3 activation and cell death. Thus, inhibition of the mitochondrial NCLX in neurons with misfolded or mutated tau can be involved in the mechanism of neurodegeneration.     

Moment closure for local control models of calcium-induced calcium release in cardiac myocytes

In prior work, we introduced a probability density approach to modeling local control of Ca²⁺-induced Ca²⁺ release in cardiac myocytes, where we derived coupled advection-reaction equations for the time-dependent bivariate probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum (SR) [Ca²⁺] conditioned on Ca²⁺ release unit (CaRU) state. When coupled to ordinary differential equations (ODEs) for the bulk myoplasmic and network SR [Ca²⁺], a realistic but minimal model of cardiac excitation-contraction coupling was produced that avoids the computationally demanding task of resolving spatial aspects of global Ca²⁺ signaling, while accurately representing heterogeneous local Ca²⁺ signals in a population of diadic subspaces and junctional SR depletion domains. Here we introduce a computationally efficient method for simulating such whole cell models when the dynamics of subspace [Ca²⁺] are much faster than those of junctional SR [Ca²⁺]. The method begins with the derivation of a system of ODEs describing the time-evolution of the moments of the univariate probability density functions for junctional SR [Ca²⁺] jointly distributed with CaRU state. This open system of ODEs is then closed using an algebraic relationship that expresses the third moment of junctional SR [Ca²⁺] in terms of the first and second moments. In simulated voltage-clamp protocols using 12-state CaRUs that respond to the dynamics of both subspace and junctional SR [Ca²⁺], this moment-closure approach to simulating local control of excitation-contraction coupling produces high-gain Ca²⁺ release that is graded with changes in membrane potential, a phenomenon not exhibited by common pool models. Benchmark simulations indicate that the moment-closure approach is nearly 10,000-times more computationally efficient than corresponding Monte Carlo simulations while leading to nearly identical results. We conclude by applying the moment-closure approach to study the restitution of Ca²⁺-induced Ca²⁺ release during simulated two-pulse voltage-clamp protocols.     

Study on the relationship between calcium-induced calcium release from mitochondria and PTP opening

The permeability transition pore (PTP) is central for mitochondria function. PTP either open in low-conductance state to carry out mCICR (Ca²⁺-induced Ca²⁺ release from mitochondria) and play roles in cell phsyiological activities or open in high-conductance conformation to release harmful substances and play important roles in cell pathological responses and apoptosis. The results of study on the relationship between mCICR and PTP opening show Ca²⁺ concentrations but not the Ca²⁺ delivery mode determined the occurrence of mCICR or PTP opening. Ca²⁺-induced PTP opening began with and depended on mCICR. mCICR was a prerequisite for H₂O₂ and As₂O₃-induced PTP opening. The results indicated that the PTP opening was determined by Ca²⁺ stimulation intensity but not mode. PTP could switch from low- to high-conductance conformation and the PTP open by high-conductance began with low-conductance state. mCICR is necessary for Ca²⁺-dependent PTP opening. Our data suggested also that it would be possible to control cellular responses first by modulating mCICR, then by regulating PTP opening.     

Cyclic ADP-ribose and the regulation of calcium-induced calcium release in eggs and cardiac myocytes

Cyclic ADP-ribose (cADPR) is a cyclic metabolite of NAD⁺ synthesised in cells and tissues expressing ADP-ribosyl cyclases. Although it was first discovered in sea-urchin egg extracts as a potent calcium mobilizing agent, subsequent studies have indicated that it may have a widespread action in the activation of calcium-release channels in such diverse systems as mammalian neurones, myocytes, blood cells, eggs, and plant microsomes. In this review we focus on recent work suggesting that cADPR enhances the sensitivity of ryanodine-sensitive calcium-release channels (RyRs) to activation by calcium, a phenomenon termed calcium-induced calcium release (CICR). Two roles for cADPR in calcium signaling are discussed. The first is as a classical second messenger where its levels are controlled by extracellular stimuli, and the second mode of cellular regulation is that the levels of intracellular cADPR may set the sensitivity of RyRs to activation by an influx of calcium in excitable cells. These two possible actions of cADPR are illustrated by considering the signal transduction events during the fertilization of the sea-urchin egg and the modulation of CICR during excitation-coupling in isolated guinea-pig ventricular myocytes, respectively.     

Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes

It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine- and depolarization-induced calcium responses were inhibited, whereas acetylcholine- and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeine- and depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.     

Effects of silver ion on the calcium-induced calcium release channel in isolated sarcoplasmic reticulum

Ag+-induced Ca2+ release in isolated sarcoplasmic reticulum (SR) was studied by the stopped flow method monitoring chlortetracycline fluorescence change. After improving the experimental procedure, the initial rate of Ca2+ release could be determined more precisely than before. Micromolar concentrations of Ag+ specifically enhanced Ca2+ efflux from heavy fraction of SR vesicles (HSR). This specific effect was referred to as Ag+-induced calcium release. The Ag+-induced Ca2+ efflux was activated by caffeine and ATP, but was inhibited by Mg2+ and procaine. Further, Ag+ enhanced the Ca2+-induced Ca2+ release over the whole range of Ca2+ concentrations, similarly to ATP. Parallel to Ca2+ efflux, Mg2+ efflux, measured by the same method, was also activated by Ag+. Choline permeability determined by the light scattering method was also activated by Ag+. The results suggest that Ag+ binds to the activation site of the Ca2+-induced Ca2+ release channel and opens the channel. The Ag+ binding site is different from the Ca2+ binding site but similar to the ATP binding site.     

Temperature-dependent calcium-induced calcium release via InsP3 receptors in mouse olfactory ensheathing glial cells

Cooling can induce Ca(2+) signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca(2+) signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca(2+) transients that persisted in the absence of external Ca(2+), but were suppressed after Ca(2+) store depletion by cyclopiazonic acid. Cooling-evoked Ca(2+) transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP(3) receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca(2+) transients. Moderate Ca(2+) increases, as evoked by flash photolysis of NP-EGTA (caged Ca(2+)) and cyclopiazonic acid, triggered InsP(3) receptor-mediated Ca(2+) release at 22°C, but not at 31°C. The results suggest that InsP(3) receptors mediate Ca(2+)-induced Ca(2+) release in OECs, and that this Ca(2+) release is temperature-sensitive and can be suppressed at temperatures above 28°C.     

NADH-Linked substrate-mediated enhancement of mitochondrial calcium accumulation and DNA single-strand breakage elicited by tert-butylhydroperoxide: the source of the cation is a ryanodine-sensitive calcium store.

We previously found that the membrane-permeant NADH-linked substrates pyruvate and beta-hydroxybutyrate enhance the formation of DNA single-strand breaks induced by tert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.