RyR Clusters Research Articles

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine).We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.

Live Cell Palm Techniques for Super Resolution Imaging of Murine Cardiac Myocytes

Since their inception, super resolution methodologies have revolutionised the way in which biologists can visualise subcellular structures. Cardiac science has particularly benefited under these methodologies, as structures such as the dyads of cardiomyocytes have become resolvable, thus yielding new understanding of -their spatial organization. However, pairing of nanoscale dyadic structure and function has been hampered by the fact that techniques such as direct stochastic optical reconstruction microscopy (dSTORM) generally require fixed tissue. Furthermore, quantification of dSTORM images based on antibody labelling is complicated by variability in labelling efficacy, and displacement of the fluorophore from the protein of interest. We presently circumvented these limitations by creating a transgenic mouse expressing a photo activated red fluorescent protein tagged to ryanodine receptor 2 (PA-tag-RFP-RyR2). Imaging of live, isolated cardiomyocytes was attained with a commercial Zeiss Elyra dSTORM setup using the photo-activated light microscopy (PALM) technique. PA Tag RFP requires dual sample illumination with activation laser and imaging laser to produce constant photo switching. In our experiments we found that by adjusting the rate of activation and photobleaching from imaging we could optimize the blinking behaviour required for live cell PALM imaging. To reduce background, HILO and TIRF illumination were employed to reduce the thickness of the optical section. Our results show RyR localisation accuracy of 40nm in the X and Y plane for live cell images, in contrast to the higher resolution of 10-20nm in fixed samples with antibody labelling. RyR cluster arrangements in live cells were observed to be broadly in agreement with fixed samples. Ongoing work is aimed at linking RyR organization to local calcium release events (Ca2+ sparks), with focus on unravelling the functional consequences of the dynamic nature of RyR arrangement.

Dynamic and Irregular Distribution of RyR2 Clusters in the Periphery of Live Ventricular Myocytes

Cardiac ryanodine receptors (RyR2s) are Ca2+ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2+ release. The distribution of these Ca2+ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2+ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2+ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2+- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2+ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

How Does Calcium Overload Generate Calcium Waves in Heart?

Calcium-induced calcium (Ca2+) release (CICR) is the fundamental signaling mechanism that enables the voltage-sensitive L-type Ca2+ channels (LCCs) in the sarcolemma to activate the cellular [Ca2+]i transient and thus contraction in the heart. In CICR the LCC Ca2+ influx activates nearby Ca2+ release channels (ryanodine receptors, RyR2s) across a 15 nm “subspace” gap via CICR, just as the Ca2+ released from any open RyR2 can activate other RyR2s located within the same RyR2 cluster. The junctional ensemble of LCCs and the opposing RyR2s constitute the cardiac “Ca2+ release unit” or CRU. When a CRU is activated by any means it produces a Ca2+ spark. Excitation-contraction (EC) coupling is the process by which CRUs are activated quasi-synchronously by the voltage-activated LCCs in a cell during the action potential (AP). Importantly, CICR also occurs independently when the probabilistic opening of RyR2s produce a Ca2+ spark in the absence of an LCC trigger. Ca2+ sparks and individual RyR2 openings constitute the endogenous Ca2+ leak pathway from the sarcoplasmic reticulum (SR), the “cellular Ca2+ store”. However, in pathological conditions the Ca2+ leak can become unstable and produce cell-wide Ca2+ waves and life-threatening Ca2+-dependent arrhythmias. During SR Ca2+ overload, it has been suggested that a putatively novel phenomena dubbed “Store Overload Induced Ca2+ Release” or “SOICR”, that is distinct from CICR, may develop. SOICR's distinctiveness hinges on the molecular ability of SR lumenal Ca2+ to directly activate RyR2 openings without the need for elevated Ca2+ in the subspace to produce CICR. To investigate this hypothesis, we extend our existing model of RyR2 gating to include a formulation for SOICR whereby SR Ca2+ can directly activate a RyR2 channel to open. Using a multi-scale modeling approach that spans from single channel to whole-cell and spatial simulations, we show that both CICR and SOICR gating modes can indeed activate RyR2 channels and modify Ca2+ spark dynamics in a manner consistent with experimental observations. However, detailed comparison of Ca2+ wave generation and CRU-to-CRU Ca2+ wave propagation shows that CICR alone is a sufficient, and necessary, mechanism to explain Ca2+ release in heart under both physiological and pathological conditions.

Local Recovery of CICR Probed by Ultra-Effective Calcium Uncaging

In cardiac myocytes a small amount of Ca2+ enters the cytoplasm upon depolarization and initiates Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). Ca2+ release occurs via subcellular local Ca2+ signaling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both, SR refilling and restoration of Ca2+ sensitivity of the RyRs. In this work we used two photon (2P) photolysis of the ultra effective caged Ca2+ compound BIST-2EGTA [1] and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in presence of a low dose of caffeine (to mimic sensitisation of RyRs) or cyclopiazonic acid (CPA, SERCA inhibitor to slow SR refilling). Whole-cell patch-clamped cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (5 ms, 810 nm) with different intra pulse intervals were applied to test Ca2+ release amplitude recovery. Data recorded in the presence of CPA showed slower recovery with a time constant of ∼76 ms compared to control condition (∼56 ms). In the presence of a low dose of caffeine the recovery was faster than control (∼31 ms). We obtained comparable results with the well established “ryanodine method” [2], where a low dose of ryanodine is used to trigger repetitive sparks from single cluster of RyRs. This new 2P approach provides composite information of Ca2+ release amplitude recovery reflecting both SR refilling and restoration of Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias. 1. Agarwal, H. K., Janicek, R., Chi, S.-H., Perry, J. W., Niggli, E., and Ellis-Davies, G. C. R. (2016), Calcium uncaging with visible light. JACS 138, 3687-3693. 2. Sobie, E. A., Song, L.-S. and Lederer, W. J. (2005), Local recovery of Ca2+ release in rat ventricular myocytes. J. Physiol, 565, 441-447.

Mathematical modeling shows the frequency of Ca2+ sparks in cells depends on the ryanodine receptor’s arrangement

Calcium dynamics plays key role in many intracellular processes. Studying of calcium sparks, that is events of calcium release via groups of ryanodine receptors, or RyR channels, is of a great importance for live sciences. Recent experimental studies show, that RyRs have a non-uniform arrangement in calcium release units (CRU), however, to our knowledge, there is yet no published Ca2+ spark model which takes into account this fact. In the paper we made use of a genetic simulated annealing (GSA) algorithm for the RyR cluster generation and Gillespie algorithm for modeling calcium spark. Computer simulation of the model CRU taking into account a specific homotetrameric structure of the RyR channel shows that experimental nearest neighbor distances (NND) distributions of RyRs in CRU do not provide exact information about calcium spark probability. Having the same NND distribution, the CRUs can have different inverse distance matrices and hence different calcium spark properties. Our model approach opens novel perspectives for studying the calcium release process.

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces I CaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.

168-03: Axial membrane tubules in atrial myocytes initiate rapid intracellular calcium signals through a new “super-hub” mechanism

Invaginated sarcolemmal membrane structures in cardiac myocytes form transverse tubules (TTs). The TTs form electrical and chemical signaling conduits from the myocyte exterior to regions deep in the cell such as the junctional sarcoplasmic reticulum (jSR) critical in excitation-contraction (EC) coupling. Local control of jSR Ca2+ release in ventricular myocytes (VM) depends critically on this organization. In contrast, atrial myocytes (AM) normally have few if any TTs (although the exact abundance has been controversial). In atrial myocytes, the normally sparse distribution of TTs suggests that a critical role in EC coupling remains unlikely. Here, we identify a structurally distinct and mechanistically novel role for even sparsely set TTs in atrial EC coupling. Using novel membrane-preserving live cell techniques, we identify relatively abundant axial membrane tubules (ATs). These ATs are deep intracellular branches of the relatively rare TTs in 100% of AM investigated (n = 29). Using live cell STimulated Emission Depletion (STED) superresolution microscopy, we show that atrial AT structures are characteristically different from TTs. ATs represent both the major component of the cell-wide tubule network and the largest tubules: AT circumference in AM 921 ± 24 nm vs. VM 630 ± 26 nm; p < 0.001 (physiological live cell conditions). These dimensions indicate unprecedentedly large AT surfaces with a positive membrane curvature at the AM center suggesting that ATs could function as a central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined AT and intracellular Ca2+ transient (CaT) imaging. High-amplitude CaTs originated directly from ATs creating highly localized central-axial Ca2+ signals. Importantly, local CaT onset measurements showed that atrial CaTs occurred significantly earlier at AT compared to surface membrane locations (p < 0.05). To explain the observed CaT behaviors, we imaged in situ the Ser-2808 phospho-epitope of ryanodine receptor (RyR2) Ca2+ release channels necessary for PKA phosphorylation. RyR2 channel clusters at AT-hubs showed increased PKA phosphorylation in contrast to the low phosphorylation state of non-junctional RyR2 clusters in unstimulated AM. Sarcomere shortening experiments confirmed significantly larger and faster AM compared with VM axial contractions (p < 0.05); this effect was significantly augmented by isoproterenol (1 µm) through non-junctional cluster recruitment. Consistent with the proposed AT super-hub model, genetic ablation of RyR2 PKA phosphorylation in Ser-2808-Ala knockin mice significantly attenuated atrial sarcomere shortening and in vivo atrial but not ventricular contractile function. Our data suggest a fundamentally new atrial EC-coupling model where AT structures in atrial myocytes function as large signaling super-hubs that couple central-axial Ca2+ signaling to rapid contractile activation. This supports a critical role of the transverse-axial tubular system in cell-specific atrial EC-coupling mechanisms (i.e. faster contractions than in ventricles) and questions previous models of atrial EC-coupling based on slow intracellular Ca2+ diffusion. Genetic ablation of RyR2 cluster phosphorylation unmasks a previously unknown role of atrial PKA regulation, which may explain atrial function in health versus decreased contractility occuring in atrial fibrillation and heart failure.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes

In Ca2+-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca2+ level to trigger and sustain Ca2+ waves, that propagate at constant rate (ʋwave). High luminal Ca2+ sensitizes RyR2, thereby increasing Ca2+ sparks frequency, and the larger RyR2-mediated SR Ca2+ flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca2+ reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca2+ removal could instead impair RyR2 activation. We assessed whether rapid SR Ca2+ uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca2+ waves amplitude was largely unaffected. Furthermore, SR Ca2+ also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca2+ and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca2+ determines ʋwave, while rapid SERCA Ca2+ uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca2+.

Sheet-Like Remodeling of the T-System of Ventricular Cardiomyocytes in Heart Failure

The transverse tubular system (t-system) is a membrane specialization of striated myocytes crucial for excitation-contraction coupling. Various studies demonstrated remodeling of t-system in heart disease in species including rodent, canine and human. Commonly, disease was associated with heterogeneous t-system depletion. Here, we test the hypothesis that t-system remodeling underlies inefficient excitation-contraction coupling in human heart failure.We investigated the t-system in left-ventricular myocardium samples from 5 patients undergoing cardiac surgery. The tissues were labeled for ryanodine receptors (RyRs), and the extracellular space including the t-system. We acquired 3D images with a Leica SP8 confocal microscope using a 63x oil immersion lens. After image deconvolution, we segmented individual cardiomyocytes. We reconstructed the t-system and quantified the RyR intensity and distance of RyR clusters to the t-system. Furthermore, isolated myocytes were labeled with di-8-ANEPPS and Fluo-4 to acquire 2D image sequences of calcium transients using a Zeiss Live 5 Duo confocal microscope.Our studies revealed a novel type of t-system remodeling, characterized by flat sheets rather than tubular structures. Three-dimensional reconstructions indicated that sheet cavities were connected with the extracellular space. Principal component analysis showed that sheets were aligned with the longitudinal myocyte axis. One sample did not show sheets and served as control. Comparing 5 cells with sheets to 5 control cells, we found significantly increased distances between RyR clusters and the t-system (0.82±0.12µm and 0.5±0.06µm, respectively) and a significantly higher fraction of RyRs present >1µm from the sarcolemma (49±6.9% and 21±5.5%, respectively). Cardiomyocytes with sheets exhibited spatial heterogeneity and asynchrony of calcium transients.Our studies present a novel phenotype of remodeled t-system, with a strikingly increased number of non-junctional RyR clusters. We suggest that the associated spatially heterogeneous and asynchronous calcium release contributes to reduced contractility in heart failure.

Cardiac Pacemaker Cell Function at a Super-Resolution Scale of SIM: Distribution of RyRs, Calcium Dynamics, and Numerical Modeling

Previous confocal microscopy studies of SA-node cells revealed (in tangential sections) hierarchical clustering of calcium release channels (RyRs). Further numerical model simulations demonstrated that such hierarchy can enable propagating Local-Calcium-Releases (LCRs), crucial events of pacemaker cell operation. RyR distributions in 3D remain, however, unknown, leaving substantial uncertainty in constructing realistic models of SA-node cells. Here we approached the problem by super-resolution structured illumination microscopy (SIM) achieving approximately double the resolution of conventional microscopy to measure both 3D-distribution of RyRs and 2D-calcium dynamics in isolated rabbit SA-node cells. Each 3D-distribution of RyR clusters was reconstructed with 150 nm z-sectioning to capture the entire cell (bottom-to-top). We developed a novel computer algorithm to detect and characterize fluorescence signals of RyR clusters, including their sizes (volumes) and genuine distances to their nearest neighbors in 3D. We analyzed 8 cells with RyR localization mainly at the cell surface and found from 1107 to 2420 (mean=2070) RyR clusters in each cell. Distributions for nearest neighbor (center-to-center) distance were essentially (>95%) within the range 200-800 nm (mean=405 nm) that is substantially smaller than we previously reported (mean∼800 nm), indicating that many small clusters (lacking in confocal scans) now become resolved by SIM and also many neighboring clusters previously missed in 2D-projections now become revealed by the new 3D-analysis. Our cell-mid-section SIM measurements of calcium dynamics (using fluo-4) revealed that LCRs occur mainly within ∼1µm under the cell surface, in agreement with immunofluorescence data on RyR cluster localization. The new SIM results on Ca dynamics and anatomical information are included in new numerical modeling of SA-node cell. The larger number of clusters and shorter distances facilitate calcium-induced-calcium-release and LCR propagation observed experimentally.

Axial Membrane Tubules in Atrial Cardiomyocytes Confine Ultrarapid Intracellular Calcium Signals through a New Super-Hub Mechanism

Endocytic membrane structures called transverse tubules (TTs) are thought to represent the main mechanism for contacts with the junctional sarcoplasmic reticulum (jSR) and intracellular Ca2+ release in ventricular cardiomyocytes (VCM). In contrast, in atrial cardiomyocytes (ACM) we identify previously not described large axial membrane tubules (ATs) at the cell core. Using optimized ACM isolation and live cell superresolution microscopy, we show that AT membrane structures represent both the major (ie. >90% of endocytic tubule structures) and largest tubule component (mean AT circumference 921±24 nm). Due to the unusually large AT membrane surface with a positive curvature exposed to the cytoplasm, we hypothesized that the endocytic AT membrane surface functions as central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined imaging of AT membranes with fast intracellular Ca2+ imaging. Indeed, high-amplitude Ca2+ transients originated in a highly localized fashion rapidly from the AT-hubs. Importantly, Ca2+ transients originating from AT-hubs occured significantly earlier compared to cellular surface locations (p<0.05). This suggests a new EC-coupling model where AT-hubs control rapid centrifugal Ca2+ signals. Sarcomere shortening confirmed significantly faster ACM versus VCM EC-coupling (p<0.05). Furthermore, in situ phospho-epitope imaging correlated ryanodine receptor (RyR2) channel phosphorylation with AT-hub function. Only junctional RyR2 clusters at AT-hubs showed increased phosphorylation compared to non-junctional clusters. Genetic ablation of RyR2-specific phosphoepitopes by Ala-knockin impaired ACM contractility, consistent with loss-of-function and disruption of AT-hub-dependent signaling. Hence, our data support a fundamentally new ACM model of EC-coupling, where AT-membranes function as signaling super-hubs that control high-amplitude ultrarapid Ca2+ signals at the cell core. Genetic ablation of RyR2 cluster phosphorylation further suggests that the super-hub model is relevant to explain atrial function in health and decreased contractility typical for atrial disease forms.

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes.

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as 'junctions', are fast communication hubs within excitable cells such as neurones and muscle. Here, we have examined two transgenic murine models probing the role of junctophilin-2, a membrane-tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single-molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR; in this paper, we use RyR to refer to the myocardial isoform RyR2). Although this change was associated with much enlarged RyR clusters that, due to their size, should be more excitable, functionally it caused a mild inhibition in the Ca2+ signalling output of the junctions (Ca2+ sparks). Analysis of the single-molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the Ca2+ signalling properties of the junction and thus the contractile function of the cell.

On the Adjacency Matrix of RyR2 Cluster Structures.

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca2+ channels and flux of Ca2+ into the cells. This increases Ca2+ binding to ligand-gated channels known as ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca2+, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca2+ release events known as “sparks.” RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Our recent work has shown that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca2+ sparks. We showed that the probability of a Ca2+ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. Thus, “function” follows from “structure.” This probability is related to the maximum eigenvalue (λ 1) of the adjacency matrix of the RyR2 cluster lattice. In this work, we develop a theoretical framework for understanding this relationship. We present a stochastic contact network model of the Ca2+ spark initiation process. We show that λ 1 determines a stability threshold for the formation of Ca2+ sparks in terms of the RyR2 gating transition rates. We recapitulate these results by applying the model to realistic RyR2 cluster structures informed by super-resolution stimulated emission depletion (STED) microscopy. Eigendecomposition of the linearized mean-field contact network model reveals functional subdomains within RyR2 clusters with distinct sensitivities to Ca2+. This work provides novel perspectives on the cardiac Ca2+ release process and a general method for inferring the functional properties of transmembrane receptor clusters from their structure.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release.

AimsIn atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF.Methods and resultsUsing high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks.ConclusionIn persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release.

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes.

Abnormalities in cardiomyocyte Ca2+ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca2+ sparks (brief, localized increased in [Ca2+]i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca2+]i transient amplitude and sarcoplasmic reticulum (SR) Ca2+ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca2+ sparks were less frequent, whereas the fractional release and the frequency of Ca2+ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca2+ sparks were more frequent in PMI cells (under conditions of similar SR Ca2+ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca2+ sparks frequency normalized by the SR Ca2+ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca2+ spark frequency. Indeed, using an internal "failing solution" with less ATP (as found in HF), we observed a dramatic decrease in Ca2+ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca2+ load may mask these effects at the Ca2+ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca2+ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 μM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

Simultaneous Detection and Colocalization of Calcium Sparks and Ryanodine Receptor Clusters in Cardiac Myocytes

Further understanding of calcium handling and excitation-contraction coupling in cardiac myocytes requires quantitative data analysis methods to characterize calcium release events in terms of structural properties of the cell. Such automatic methods provide a robust, consistent and reproducible characterization of physiological systems from observed experimental data. We present a multilevel analysis that simultaneously focuses on both the occurrence of spontaneous calcium sparks and the distribution of RyR clusters across a cardiac myocyte. The approach localizes the release events and determines the distance to the nearest ryanodine receptor cluster, therefore providing quantitative information on the spatio-temporal distribution of activation sites in the cell. The location of clusters takes into account motion artifacts produced by calcium waves or mini-waves. The method has been validated with line-scan confocal microscopy data from mouse ventricular myocytes and provides a full characterization of the spark morphology including its amplitude, baseline, decay time, upstroke time, Full-Width at Half-Maximum, Full-Duration at Half-Maximum and background noise.The processing is applied to linescan images of both RyR clusters and calcium fluorescence. It includes the following steps: i) Adaptive filtering of background fluorescence fluctuations using a robust estimation of the noise by means of the median absolute deviation of the basal fluorescence signal. ii) Individual sparks are detected by using a modified watershed segmentation method that includes stopping rules for both event size and shape. iii) Location of RyR clusters was implemented by thresholding a continuous wavelet transform of the cluster image (robust to motion and contraction). iV) Measurement of spark morphology properties, including the distance from each spark to the closest RyR cluster.