RyR Clusters Research Articles

Structure-Function Relationship of the Ryanodine Receptor Cluster Network in Sinoatrial Node Cells.

The rate of spontaneous action potentials (APs) generated by sinoatrial node cells (SANC) is regulated by local Ca2+ release (LCR) from the sarcoplasmic reticulum via Ca2+ release channels (ryanodine receptors, RyRs). LCR events propagate and self-organize within the network of RyR clusters (Ca release units, CRUs) via Ca-induced-Ca-release (CICR) that depends on CRU sizes and locations: While larger CRUs generate stronger release signals, the network's topology governs signal diffusion and propagation. This study used super-resolution structured illumination microscopy to image the 3D network of CRUs in rabbit SANC. The peripheral CRUs formed a spatial mesh, reflecting the cell surface geometry. Two distinct subpopulations of CRUs were identified within each cell, with size distributions conforming to a two-component Gamma mixture model. Furthermore, neighboring CRUs exhibited repulsive behavior. Functional properties of the CRU network were further examined in a novel numerical SANC model developed using our experimental data. Model simulations revealed that heterogeneities in both CRU sizes and locations facilitate CICR and increase the AP firing rate in a cooperative manner. However, these heterogeneities reduce the effect of β-adrenergic stimulation in terms of its relative change in AP firing rate. The presence of heterogeneities in both sizes and locations allows SANC to reach higher absolute AP firing rates during β-adrenergic stimulation. Thus, the CICR facilitation by heterogeneities in CRU sizes and locations regulates and optimizes cardiac pacemaker cell operation under various physiological conditions. Dysfunction of this optimization could be a key factor in heart rate reserve decline in aging and disease.

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.

Cardiomyocyte ryanodine receptor clusters expand and coalesce after application of isoproterenol.

Earlier work has shown that ventricular ryanodine receptors (RyR2) within a cluster rearrange on phosphorylation as well as with a number of other stimuli. Using dSTORM, we investigated the effects of 300 nmol/liter isoproterenol on RyR2 clusters. In rat ventricular cardiomyocytes, there was a symmetrical enlargement of RyR2 cluster areas, a decrease in the edge-to-edge nearest neighbor distance, and distribution changes that suggested movement to increase the cluster areas by coalescence. The surface area covered by the phosphorylated clusters was significantly greater than in the control cells, as was the cluster density. This latter change was accompanied by a decreased cluster fragmentation, implying that new tetramers were brought into the sarcoplasmic reticulum. We propose a possible mechanism to explain these changes. We also visualized individual RyR2 tetramers and confirmed our earlier electron-tomographic finding that the tetramers are in a disorganized but non-random array occupying about half of the cluster area. Multiclusters, cluster groups defined by the maximum distance between their members, were analyzed for various distances. At 100 nm, the areas occupied by the multiclusters just exceeded those of the single clusters, and more than half of the multiclusters had only a single subcluster that could initiate a spark. Phosphorylation increased the size of the multiclusters, markedly so for distances >100 nm. There was no relationship between the number of subclusters in a group and the area covered by it. We conclude that isoproterenol induces rapid, significant, changes in the molecular architecture of excitation-contraction coupling.

Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices.

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death.

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Thyroid hormone maintains cardiac dyadic structures by regulating junctophilin-2 localization to ryanodine clusters

Overt and subclinical hypothyroidism are associated with increased risk of cardiovascular disease and mortality. In pre-clinical studies, supplementation with triiodo-L-thyronine (T3) has been shown to regulate cardiomyocyte T-tubule (TT) organization, and thereby maintain ryanodine receptor-2 and L-type Ca 2+ channel (RyR-LTCC) clusters in dyadic structures necessary for efficient excitation-contraction (EC) coupling. We hypothesized that the TT-sarcoplasmic reticulum (SR)-spanning protein Junctophilin-2 (Jph) is regulated by T3. Thyroid hormone (TH) deficiency was induced in 12 adult female Sprague-Dawley rats by oral propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU treatment or with T3 (10 μg/kg/day) supplementation for 2 weeks (PTU+T3). Untreated rats served as euthyroid (EU) controls. Thyroid status was confirmed by plasma T3 and T4 levels. Left ventricular cardiomyocytes (CM) were isolated by collagenase digestion for single molecule imaging of Jph2, RyR2 and LTCC proteins using 2D and 3D STORM (stochastic optical reconstruction microscopy; Oxford Nanoimager). DBSCAN algorithms for cluster analysis (Web-based programs developed by LumeVR) measured cluster size and number, nearest neighbor cluster distances, and protein co-localizations. Calcium sparks in live CMs loaded with Fluo-4 were captured by line-scanning laser microscopy (Zeiss 980) to assess spontaneous SR Ca 2+ leak. STORM imaging of normal CMs showed that ~60% of RyR clusters consisted of 10-50 RyR localizations (locs) with 100-200 Jph molecules co-localized within 210 nm radius of the cluster centroid. Although Jph locs per cluster increased proportionately with increased cluster size, PTU cells had markedly less Jph co-localized with RyR at all cluster sizes compared to EU and PTU+T3 cells. The reduction of associated Jph was most evident in super-clusters (>250 RyR locs). 3D image analysis showed nearest neighbor distances between RyR clusters that were significantly increased in the PTU CMs vs EU (749±146 nm vs 640±140 nm), which were normalized with T3-treatment (616±60 nm). Numbers of RyR clusters without associated Jph locs were significantly higher in PTU cells, and reduced with T3. RyR Ca 2+ leak as measured by spontaneous Ca 2+ sparks was increased in PTU CMs, but decreased significantly to control values in T3-treated cells. This study supports a regulatory role of T3 in maintaining TT-SR dyad structures, in part by increasing Jph co-localization with RyR clusters, thus promoting efficient interaction of RyR with LTCC channels for effective EC-coupling and cardiac output. Study funded by NIH R15HL15406 and by resources of the NYIT Imaging Center. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Correlative super-resolution analysis of cardiac calcium sparks and their molecular origins in health and disease.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Super-resolved SIM imaging revealed hierarchical organization of the ryanodine receptor network in 3 dimensions in rabbit sinoatrial node cells.

The heartbeat is initiated by specialized sinoatrial node cells (SANCs). The rate of the heartbeat is regulated by Ca released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacting with voltage and Ca-dependent membrane ion currents in SANCs. The released Ca takes the form of localized Ca releases (LCRs) that propagate for limited distances in wave-like manner. A computational model of SANCs, previously developed in our laboratory, simulates LCR synchronization mechanism as hierarchical clustering of surface RyRs based on confocal microscopy imaging data obtained in tangential sections of SANCs. The true detailed distribution of RyR clusters in 3D, however, remains unknown. Here, we applied a series of new computational approaches to experimental data obtained by super-resolved structured illumination microscopy in rabbit SANCs, in which RyRs were labeled by specific antibodies. Our approach consists of automated segmentation of RyR clusters with globally optimal detection and segmentation of objects with constant internal intensity distribution, followed by object-based colocalization analysis in 3D. Results are further refined with watershed transformations and computational geometry. Examinations of the images have revealed a hierarchical architecture of RyR network (positions and sizes of submembrane RyR clusters). After constructing a mathematical convex hull around the cell using RyR clusters as nodes, clusters were grouped based on their volume. The distribution of RyR clusters exhibited a wide range of sizes from 0.75 × 10−3 µm3 to 0.50 µm3 and cluster histograms followed a Poisson-like distribution. These new data have been incorporated in a new, realistic 3D model of SANC, in which heterogeneous RyR clusters facilitate LCR propagation. Impairment of the complex, hierarchical structure of RyR clusters may lead to sinus node dysfunction in aging and disease.

InsP3R-RyR Ca2+ channel crosstalk facilitates arrhythmias in the failing human ventricle.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R-RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R-RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

Three-dimensional visualization of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads.

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery of intracellular calcium signalling in cardiomyocytes. While a range of optical super-resolution microscopy techniques have revealed the nanoscale structure of these clusters, the three-dimensional (3D) nanoscale topologies of the clusters have remained mostly unresolved. In this paper, we demonstrate the exploitation of molecular-scale resolution in enhanced expansion microscopy (EExM) along with various 2D and 3D visualization strategies to observe the topological complexities, geometries and molecular sub-domains within the RyR clusters. Notably, we observed sub-domains containing RyR-binding protein junctophilin-2 (JPH2) occupying the central regions of RyR clusters in the deeper interior of the myocytes (including dyads), while the poles were typically devoid of JPH2, lending to a looser RyR arrangement. By contrast, peripheral RyR clusters exhibited variable co-clustering patterns and ratios between RyR and JPH2. EExM images of dyadic RyR clusters in right ventricular (RV) myocytes isolated from rats with monocrotaline-induced RV failure revealed hallmarks of RyR cluster fragmentation accompanied by breaches in the JPH2 sub-domains. Frayed RyR patterns observed adjacent to these constitute new evidence that the destabilization of the RyR arrays inside the JPH2 sub-domains may seed the primordial foci of dyad remodelling observed in heart failure.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks.

Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization and function of ryanodine receptors (RyRs) in cardiomyocytes. Recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca2+ spark generation in failing cardiomyocytes is likely complex and these observations suggest that both the nanoscale organization of RyRs and the pattern of phosphorylated RyRs within clusters could be critical determinants of Ca2+ spark dynamics. To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca2+ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca2+ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca2+ dynamics in HF.

Saltatory Ca2+-induced-Ca2+ release propagation among RyR2 clusters in sinoatrial node cells isolated from GFP-targeted RYR2 mice

Rhythmic heart excitations are generated by sinoatrial node cells (SANC) operating as a coupled-clock system: an ion channel membrane clock couples to sarcoplasmic reticulum Ca2+clock that generates diastolic local Ca2+releases (LCRs) via release channels RyR2. In contrast to stochastic Ca2+sparks in ventricular myocytes, LCRs are roughly periodic at physiological [Ca2+] due to partial phosphorylation (PKA/CaMKII) of Ca2+ cycling proteins in the basal state, i.e. in the absence of beta-adrenergic-receptor stimulation.

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3′,5′-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Nanoscale Organization, Regulation, and Dynamic Reorganization of Cardiac Calcium Channels.

The architectural specializations and targeted delivery pathways of cardiomyocytes ensure that L-type Ca2+ channels (CaV1.2) are concentrated on the t-tubule sarcolemma within nanometers of their intracellular partners the type 2 ryanodine receptors (RyR2) which cluster on the junctional sarcoplasmic reticulum (jSR). The organization and distribution of these two groups of cardiac calcium channel clusters critically underlies the uniform contraction of the myocardium. Ca2+ signaling between these two sets of adjacent clusters produces Ca2+ sparks that in health, cannot escalate into Ca2+ waves because there is sufficient separation of adjacent clusters so that the release of Ca2+ from one RyR2 cluster or supercluster, cannot activate and sustain the release of Ca2+ from neighboring clusters. Instead, thousands of these Ca2+ release units (CRUs) generate near simultaneous Ca2+ sparks across every cardiomyocyte during the action potential when calcium induced calcium release from RyR2 is stimulated by depolarization induced Ca2+ influx through voltage dependent CaV1.2 channel clusters. These sparks summate to generate a global Ca2+ transient that activates the myofilaments and thus the electrical signal of the action potential is transduced into a functional output, myocardial contraction. To generate more, or less contractile force to match the hemodynamic and metabolic demands of the body, the heart responds to β-adrenergic signaling by altering activity of calcium channels to tune excitation-contraction coupling accordingly. Recent accumulating evidence suggests that this tuning process also involves altered expression, and dynamic reorganization of CaV1.2 and RyR2 channels on their respective membranes to control the amplitude of Ca2+ entry, SR Ca2+ release and myocardial function. In heart failure and aging, altered distribution and reorganization of these key Ca2+ signaling proteins occurs alongside architectural remodeling and is thought to contribute to impaired contractile function. In the present review we discuss these latest developments, their implications, and future questions to be addressed.

β2-adrenergic stimulation potentiates spontaneous calcium release by increasing signal mass and co-activation of ryanodine receptor clusters.

It is unknown how β-adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)-tagged RyR2 clusters. Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Spontaneous calcium release from single RyR2 clusters induced 91.4%±2.0% of all calcium sparks while 8.0%±1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two- than one-cluster sparks with a median and interquartile range for the cumulative distribution of 15.7±20.1 vs 7.6±5.7 a.u. (P<.01). β2-adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two- and three-cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9±23.4 a.u. vs 10.7±10.9; P=.015). Most sparks are caused by activation of a single RyR2 cluster at baseline while β-adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation.

Triiodothyronine maintains cardiac transverse-tubule structure and function

Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins.Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 μg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing.The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].