Ca Release Channel Research Articles

Elementary intracellular Ca signals approximated as a transition of release channel system from a metastable state.

Cardiac muscle contraction is initiated by an elementary Ca signal (called Ca spark) which is achieved by collective action of Ca release channels in a cluster. The mechanism of this synchronization remains uncertain. We approached Ca spark activation as an emergent phenomenon of an interactive system of release channels. We constructed a weakly lumped Markov chain that applies an Ising model formalism to such release channel clusters and probable open channel configurations and demonstrated that spark activation is described as a system transition from a metastable to an absorbing state, analogous to the pressure required to overcome surface tension in bubble formation. This yielded quantitative estimates of the spark generation probability as a function of various system parameters. We performed numerical simulations to find spark probabilities as a function of sarcoplasmic reticulum Ca concentration, obtaining similar values for spark activation threshold as our analytic model, as well as those reported in experimental studies. Our parametric sensitivity analyses also showed that the spark activation threshold decreased as Ca sensitivity of RyR activation and RyR cluster size increased.

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondria Ca uptake and buffering. Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knock out (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. An inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Our results suggest that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce Ca waves and cardiac arrhythmias in CPVT.

The role of mitochondrial-associated-membranes in Ca-dependent arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological and genetic approaches were employed to disrupt or promote MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, and increased RyR2-VDAC interactions. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. When treated with colchicine to induce a partial disruption of MAMs, arrhythmogenic Ca waves were exacerbated in intact Cnull cells. In permeabilized cells, colchicine also increased the frequency of Ca waves and reduced mitochondrial Ca uptake. Manipulation of MAMs by genetically targeting Mitofusin 2 also affected the frequency of Ca waves. Our results suggest that SR-mitochondria tethering or MAMs shape intracellular Ca signaling to impact arrhythmogenesis. Targeting this signaling microdomain may represent a novel strategy to reduce Ca waves and Ca-dependent arrhythmias.

Modeling Calcium Cycling in the Heart: Progress, Pitfalls, and Challenges.

Intracellular calcium (Ca) cycling in the heart plays key roles in excitation-contraction coupling and arrhythmogenesis. In cardiac myocytes, the Ca release channels, i.e., the ryanodine receptors (RyRs), are clustered in the sarcoplasmic reticulum membrane, forming Ca release units (CRUs). The RyRs in a CRU act collectively to give rise to discrete Ca release events, called Ca sparks. A cell contains hundreds to thousands of CRUs, diffusively coupled via Ca to form a CRU network. A rich spectrum of spatiotemporal Ca dynamics is observed in cardiac myocytes, including Ca sparks, spark clusters, mini-waves, persistent whole-cell waves, and oscillations. Models of different temporal and spatial scales have been developed to investigate these dynamics. Due to the complexities of the CRU network and the spatiotemporal Ca dynamics, it is challenging to model the Ca cycling dynamics in the cardiac system, particularly at the tissue sales. In this article, we review the progress of modeling of Ca cycling in cardiac systems from single RyRs to the tissue scale, the pros and cons of the current models and different modeling approaches, and the challenges to be tackled in the future.

Abstract 13377: Modulating Mitochondrial Phosphate Transport Shapes Intracellular Ca Signaling to Impact Arrhythmogenesis

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. Hypothesis: We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondrial Ca uptake and buffering. Methods: Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knockout (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. Results: In the presence of β agonist isoproterenol, an inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Conclusions: Modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce arrhythmogenic Ca waves and cardiac arrhythmias in CPVT.

Abstract 13523: The Role of Mitochondrial-Associated-Membranes in Ca-Dependent Arrhythmias

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. Hypothesis: We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Methods: Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological approach was employed to disrupt MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. Results: SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, as well as increased RyR2-VDAC interactions by proximity ligation assay. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. Colchicine was employed to induce a partial disruption of MAMs in Cnull cells. In intact Cnull cells perfused with β agonist isoproterenol, colchicine exacerbated arrhythmogenic Ca waves. In permeabilized cells, colchicine increased the frequency of arrhythmogenic Ca waves and reduced mitochondrial Ca uptake. Conclusions: Our results support that MAMs are promoted in CPVT cells to facilitate SR-mitochondria Ca transfer so mitochondria can function as a protective Ca buffer. Pharmacological disruption of SR-mitochondria tethering limits SR-mitochondria Ca transfer, thus exacerbating cellular arrhythmic burden.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje–myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

SR-Mitochondria Crosstalk Shapes Ca Signalling to Impact Pathophenotype in Disease Models Marked by Dysregulated Intracellular Ca Release.

Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signalling. A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose-fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake, and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria's function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD, respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs. FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodelling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ up-regulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs. FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2. In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodelling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signalling.

S-Palmitoylation of junctophilin-2 is critical for its role in tethering the sarcoplasmic reticulum to the plasma membrane

Junctophilins (JPH1-JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM). These ER/SR-PM junctions bring Ca-release channels in the ER/SR and Ca as well as Ca-activated K channels in the PM to within 10-25 nm. Such proximity is critical for excitation-contraction coupling in muscles, Ca modulation of excitability in neurons, and Ca homeostasis in nonexcitable cells. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD). Their N-terminal Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH-PM binding is not clear. We investigate whether S-palmitoylation of cysteine (Cys), a critical mechanism controlling peripheral protein binding to PM, occurs in JPHs. We focus on JPH2 that has four Cys residues: three flanking the MORN motifs and one in the TMD. Using palmitate-alkyne labeling, Cu(I)-catalyzed alkyne-azide cycloaddition reaction with azide-conjugated biotin, immunoblotting, proximity-ligation-amplification, and various imaging techniques, we show that JPH2 is S-palmitoylatable, and palmitoylation is essential for its ER/SR-PM tether function. Palmitoylated JPH2 binds to lipid-raft domains in PM, whereas palmitoylation of TMD-located Cys stabilizes JPH2's anchor in the ER/SR membrane. Binding to lipid-raft domains protects JPH2 from depalmitoylation. Unpalmitoylated JPH2 is largely excluded from lipid rafts and loses the ability to form stable ER/SR-PM junctions. In adult ventricular myocytes, native JPH2 is S-palmitoylatable, and palmitoylated JPH2 forms distinct PM puncta. Sequence alignment reveals that the palmitoylatable Cys residues in JPH2 are conserved in other JPHs, suggesting that palmitoylation may also enhance ER/SR-PM tethering by these proteins.

Abnormal Calcium Leak from Cardiac Sarcoplasmic Reticulum: New Insights Offered by Statistical Physics

Heart muscle contraction is activated by a synchronized systolic Ca release from sarcoplasmic reticulum (SR) via Ca sparks. In disease, Ca sparks fail to terminate, causing a diastolic Ca leak that decreases contraction amplitude and increases the risk of arrhythmia. The mechanisms and treatment of the abnormal Ca leak remain unclear. We have recently shown that spark termination emerges as collective behavior (synchronized closings) of Ca release channels (RyRs) that is identical to synchronization of spin orientation in ferromagnets described by a phase transition in Ising model. We employed the Ising approach to investigate and classify mechanisms of spark termination failure and Ca leak. Methods and Results: The key parameters determining whether spark termination succeeds or fails are SR Ca and RyR opening/closing rates. They define analogues of magnetic field h and the inverse temperature (β) in Ising model. Sparks terminate via a phase transition known as “h polarity reversal” and the leak emerges when h fails to change its sign. This happens when the SR depletes insufficiently and RyR openings remained partially synchronized via Ca-induced-Ca-release, generating long-lasting sparks. Leak can also occur via β, known as Onsager's “order-disorder” transition. This happens at low SR Ca, reducing RyR current and RyRs interactions, resulting in independent RyR openings. The disorder leak is distinguished from synchronized leak by larger Peierls contour lengths reflecting degree of disorder. Abnormal leak results from either a probability imbalance during synchronized firing of RyRs or from disordered RyR firing. Each leak type requires different and balanced treatment to shift RyR operation towards normal spark termination.

Redox Dependent Modifications of Ryanodine Receptor: Basic Mechanisms and Implications in Heart Diseases.

Heart contraction vitally depends on tightly controlled intracellular Ca regulation. Because contraction is mainly driven by Ca released from the sarcoplasmic reticulum (SR), this organelle plays a particularly important role in Ca regulation. The type two ryanodine receptor (RyR2) is the major SR Ca release channel in ventricular myocytes. Several cardiac pathologies, including myocardial infarction and heart failure, are associated with increased RyR2 activity and diastolic SR Ca leak. It has been suggested that the increased RyR2 activity plays an important role in arrhythmias and contractile dysfunction. Several studies have linked increased SR Ca leak during myocardial infarction and heart failure to the activation of RyR2 in response to oxidative stress. This activation might include direct oxidation of RyR2 as well as indirect activation via phosphorylation or altered interactions with regulatory proteins. Out of ninety cysteine residues per RyR2 subunit, twenty one were reported to be in reduced state that could be potential targets for redox modifications that include S-nitrosylation, S-glutathionylation, and disulfide cross-linking. Despite its clinical significance, molecular mechanisms of RyR dysfunction during oxidative stress are not fully understood. Herein we review the most recent insights into redox-dependent modulation of RyR2 during oxidative stress and heart diseases.

CD38: New target in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disease due to the lack of dystrophin, a subsarcolemmal protein, which leads to muscle plasmatic membrane fragility. The patients suffer from a progressive weakness and degeneration (necrosis) accompanied by inflammation of skeletal and cardiac muscles. The main cause of patient death, in DMD adults, is due to pathological cardiac phenotype, leading to severe dilated cadiomyopathy and heart failure. One key mechanism leading to muscle cells degeneration is linked to high cytosolic Ca2+ concentration observed in the DMD muscle cells, including cardiomyocytes. In this sense, several studies have shown that ryanodine receptors (RyRs), the Ca release channels of the sarcoplasmic reticulum are leaky in the mdx mouse, the main DMD rodent model, and generating the abnormal RyRs spontaneous Ca2+ activity in DMD. We propose to target this Ca2+ homeostasis deregulation, and particularly the RyRs leak, by inhibiting CD38, an enzyme which produces two Ca2+ releasing messengers: cADPR and NAADP. cADPR activates RyRs and NAADP recruits the endolysosomal calcium store, and it has also been proposed to enhance the RyRs sensitivity through Ca2+-induced Ca2+ release. In order to test this hypothesis, we evaluated the cardiac benefic effects of CD38 deletion by generating an mdx/CD38−/− mouse. We measured the RyRs spontaneous Ca2+ activity and sensitivity with Ca2+ imaging (confocal fluorescence microscopy) in isolated cardiomyocytes, the heart necrosis with Masson's trichrome staining, and the cardiac function by echocardiography. We show that CD38 deletion in the mdx completely normalized the pathological spontaneous Ca2+ activity in cardiomyocytes, reduced the heart necrosis and normalized the left ventricle dilatation and the left ventricular ejection fraction, both impaired in the mdx mouse. We propose here an innovative strategy by targeting a Ca2+ homeostasis modulator, CD38, what could lead to new therapeutic perspectives.

Positive Feedback Mechanisms among Local Ca Releases, NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (ICaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to ICaL activation threshold. During the ignition phase, ICaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and ICaL, as diastolic LCRs begin to occur below and continue within the voltage range of ICaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.

Deciphering Functions of Intracellular Formaldehyde: Linking Cancer and Aldehyde Metabolism.

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.

Stabilization of diastolic calcium signal via calcium pump regulation of complex local calcium releases and transient decay in a computational model of cardiac pacemaker cell with individual release channels.

Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (<100 nM) at high Pup, but remained elevated during slower decay at low Pup. Release propagation is facilitated at higher Pup by a larger LCR amplitude, whereas at low Pup by higher background Ca. While at low Pup LCRs show smaller amplitudes, their larger durations and sizes combined with longer transient decay stabilize integrals of diastolic Ca and NCX current signals. Thus, the local interplay of SR Ca pump and release channels regulates LCRs and Ca transient decay to insure fail-safe pacemaker cell operation within a wide range of rates.

Clusters of calcium release channels harness the Ising phase transition to confine their elementary intracellular signals

Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal-to-noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters (release units) containing a few to several hundred release channels. The channels synchronize their openings via Ca-induced Ca release, generating high-amplitude local Ca signals known as puffs in neurons and sparks in muscle cells. Despite the positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. Our simple quantitative criterion closely predicts the Ca store depletion level required for spark termination for each cluster size. We further formulate exact requirements that a cluster of release channels should satisfy in any cell type for our mapping to the Ising model and the associated formula to remain valid. Thus, 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. Our results provide exact mapping of a nanoscale biological signaling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology.

Increased Density of SERCA Pumps at the Periphery of Cardiac Purkinje Cells after Myocardial Infarction

BACKGROUND. The trigger of ventricular tachyarrhythmias after myocardial infarction (MI) has been attributed to increased incidence of electrogenic Ca2+ waves in cardiac Purkinje cells (Pcells). A change in Ca2+ release channel (CRC) gating properties and overload of intracellular Ca2+ store are 2 potential causes of this increase. We analysed through a model of intracellular Ca2+ dynamics the spontaneous Ca2+ transients of Pcells after MI. Regional protein expression was assessed for potential remodelling associated with ischemia.METHODS. Pcells were dispersed enzymatically from dog, pig, sheep, and human Purkinje fibers dissected from normal hearts and from hearts with MI. Spontaneous local Ca2+ transients were studied by high resolution confocal fluorescence microscopy techniques. Specific antibodies were used to map proteins involved in Ca2+ release/uptake functions (ERCs, SERCAs).RESULTS. In Pcells of MI heart, the density and firing rate of Ca2+-release sites were increased by 20% [Normal: 3.0 ± 0.3 sites /25µm (14 scans; 12 cells) vs MI: 3.6 ± 0.2 sites /25µm (9 cells); P<0.05] and 120% [Normal:0.77 ± 0.10 sparks/s (42 sites, 12 cells) vs MI: 1.74 ± 0.23 sparks /s (40 sites, 9 cells); P<0.05), respectively. A model fit of Ca2+-waves in MI Pcells revealed 50% increase in amplitude, 25% faster decay, and 35% elevation of Ca2+-uptake rate. No change was observed in CRC expression. However SERCAs redistribution to the cell periphery was detected in all animal MI models and in human heart with large MI. Peripheral protein expression and gene expression of SERCA2 showed 4.5 and 2.3 times increases post MI, respectively. Interestingly, no change was observed in regulatory protein phospholamban.CONCLUSION. An unknown factor associated with myocardial ischemia mediates a dramatic increase in SERCA expression at the Pcell periphery, i.e. wherein the CRCs IP3R and RyR3 are specifically located. We propose a mechanistic model of intracellular Ca2+ cycling demonstrating that augmentation of peripheral SERCA activity is sufficient to promote pro-arrhythmic spontaneous Ca2+ release in Pcells of post-MI human heart.

Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2 + release system of Purkinje fibers in large mammalian heart

BackgroundThree distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels. MethodsGene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity. ResultsRyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3μM) and xestospongin C (XeC; 50μM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5–10μm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10μM). The velocity of wavelets was two times larger (86±16μm/s; n=14) compared to CWWs' (46±10μm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150μm/s (147±34μm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24μm/s; n=5) in the central region wherein predominant RyR2 expression was detected. ConclusionWe identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Effect of sphingosine-1-phosphate on L-type calcium current and Ca(2+) transient in rat ventricular myocytes.

Modulation of Ca(2+) homoeostasis in cardiac myocytes plays a major role in beat-to-beat regulation of heart function. Previous studies suggest that sphingosine-1-phosphate (S1P), a biologically active sphingomyelin metabolite, regulates Ca(2+) handling in cardiac myocytes, but the underlying mechanism is unclear. In the present study, we tested the hypothesis that S1P-induced functional alteration of intracellular Ca(2+) handling includes the L-type calcium channel current (ICa,L) via a signalling pathway involving P21-activated kinase 1 (Pak1). Our results show that, in rat ventricular myocytes, S1P (100nM) does not affect the basal activity of ICa,L but is able to partially reverse the effect of the β-adrenergic agonist Isoproterenol (ISO, 100nM) on ICa,L. S1P (25nM) also significantly prevents ISO (5nM)-induced Ca(2+) waves and diastolic Ca(2+) release in these cells. Our further molecular characterisation demonstrates that Pak1 activity is increased in myocytes treated with S1P (25nM) compared with those myocytes without treatment of S1P. By immunoprecipitation we demonstrate that Pak1 and protein phosphatase 2A (PP2A) are associated in ventricular tissue indicating their functional interaction. Thus the results indicate that S1P attenuates β-adrenergic stress-induced alteration of intracellular Ca(2+) release and L-type Ca(2+) channel current at least in part via Pak1-PP2A-mediated signalling.