Distribution Of Ryanodine Receptors Research Articles

3D spatial modeling of sinoatrial node cells reveals a critical role of subcellular ryanodine receptor distribution in pacemaker automaticity.

Ryanodine receptors (RyRs) represent a central component in sinoatrial node (SAN) pacemaker system, as dysregulated RyRs associated with catecholaminergic polymorphic ventricular tachycardia are linked with SAN dysfunction. Within each SAN cell (SANC), the subcellular distribution pattern of RyRs is region-dependent, varying from central SAN (mostly underneath the surface sarcolemma) to peripheral SAN (striated pattern). A similar distribution pattern is also reported for the PKA- and/or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of RyRs. The impact and functional implication of their distribution patterns on the SANC pacemaker activity have not been fully elucidated. Here, we employed our recently developed 3D spatial model of mouse SANCs to investigate how the subcellar distribution and phosphorylation of RyRs affect spontaneous firing. Our model that features a high surface-to-center gradient of RyR expression well recapitulates the coupled-clock mechanisms, and the biomarkers of simulated spontaneous action potentials agree well with those from previous experimental studies. When abolishing the gradient without changing the whole-cell expression of RyRs in the SANCs, the model predicts 15.0% slowing of the firing rate. Reversing the surface-to-center gradient of RyR distribution (i.e., higher density in the cell center vs surface sarcolemma) further reduced the firing rate (by 25.3%) while increasing the beat-to-beat variation in cycle length, due to disrupted coupling between local Ca2+ releases and the membrane clock. Likewise, preventing RyR phosphorylation caused a substantial slowing in the firing rate (17.6%) while also augmenting beat-to-beat variability. Our simulations mechanistically uncover the functional significance of subcellar RyR distribution and its phosphorylation in maintaining robust automaticity of SANCs. The modeling framework will be applied to systematically delineate the impact of Ca2+-clock protein phosphorylation, distribution, and their interplay with the membrane-clock proteins on the SANC automaticity.

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.

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

Superresolution imaging techniques have provided a better understanding of the relationship between nanoscale organization and function of the ryanodine receptors (RyRs) and, consequently, the impact of organization on calcium spark properties in heart failure (HF). While hyperphosphorylation of RyRs is generally observed during HF, the impact of RyR phosphorylation on calcium (Ca2+) sparks remains incompletely understood because several experimental studies show seemingly contradictory results.

In silico simulations reveal that RYR distribution affects the dynamics of calcium release in cardiac myocytes.

The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca2+ and Mg2+ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.

Роль рианодиновых и IP 3 -рецепторов в генерации кальциевых ответов, вызываемых трициклическими антидепрессантами в нейронах неокортекса крысы

Tricyclic antidepressants, in particular amitriptyline (ATL) and desipramine (DES), are currently used to treat depression and chronic pain of various origins, in which NMDA receptor dysfunctions play an important role. The effect of therapeutic concentrations of ATL on calcium-dependent desensitization of NMDA receptors, driven by the level of free calcium in the cytoplasm, is well-known. In addition, in cardiomyocytes, ATL and DES can cause the release of calcium into the cytoplasm from intracellular stores by opening inositol-3-phosphate (IP3R) and / or ryanodine receptors (RyR) channels. The aspect of the effect of these drugs on neurons remains poorly understood. We studied the dependence of the calcium response to DES and ATL on the activation of IP3R and RyR of the endoplasmic reticulum and mitochondria using rat neocortex neurons in primary culture. Short-term (30 s) paired (5 min interval) applications of 200 μM DES or 200 μM ATL induce similar magnitude calcium responses in cortical neurons. The use of RyR and IP3R antagonists showed that responses to ATL are blocked by the IP3R antagonist 2-APB (100 μM), while responses to DES are blocked by ryanodine, the RyR antagonist (100 nM). Since intracellular distribution of RyR and IP3R is not homogenous, it can be assumed that DES and ATL stimulate calcium release from different calcium depots, representing segments of the reticulum or mitochondria. In addition, ATL and DES, being channel blockers of NMDA receptors, inhibited calcium entry from outside the cell via activated NMDA receptors. Considering the high concentrations of DES and ATL (more than 100 μM) required to stimulate the release of deposited calcium in neurons, it seems unlikely that such effects participate their therapeutic action. However, the found specificity of DES and ATL for RyR and IP3R, respectively, can be used as a tool for experimental purposes.

Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification.

The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca2+ sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.

Effects of Varying Transverse and Axial Tubules in a Three-Dimensional Model of Calcium Signaling in the Human Atrial Myocyte

T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in ventricular myocytes. Atrial myocytes are generally thought to possess sparse irregular transverse-tubular (TT) components, as opposed to the highly dense and regular ventricular TT system. Axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum that include ryanodine receptor (RyR) clusters, characterized by rapid activation of Ca2+-induced Ca2+ release, have also been identified in atrial myocytes. AT and TT remodeling and changes in distribution, composition, and phosphorylation status of Ca2+ release units are thought to underlie Ca2+ abnormalities in atrial fibrillation (AF), the most common cardiac arrhythmia. Here, we performed a computational analysis to investigate how changes in the tubular network affect human atrial electrophysiology. We modified our well-established three-dimensional model of Ca2+ signaling in the rabbit ventricular myocyte to develop an analogous model in the human atrium. We also coupled the Ca2+ signaling model to our well-established model of membrane electrophysiology in the human atrial myocyte. We systematically varied TT and AT density and RyR distribution and assessed the effect on Ca2+ spark and wave properties. When TT density is low, as shown in isolated atrial myocytes, the model recapitulates the typical U-shaped Ca2+ wave seen experimentally in transverse confocal line-scan images. Introduction of ATs resulted in W-shaped Ca2+ waves, reflecting more synchronous Ca2+ release. The frequency and amplitude of Ca2+ release events were affected by both density of TT and AT, as well as RyR distribution. Our newly developed three-dimensional model of the human atrial myocyte will be employed to investigate whether and how AF-induced cellular electrical and structural remodeling effects collectively contribute to the membrane potential and Ca2+ abnormalities seen in AF.

The Subcellular Distribution of Ryanodine Receptors and L-Type Ca2+ Channels Modulates Ca2+-Transient Properties and Spontaneous Ca2+-Release Events in Atrial Cardiomyocytes.

Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

Erratum to: Spontaneous inward currents reflecting oscillatory activation of Na+/Ca2+ exchangers in human embryonic stem cell-derived cardiomyocytes.

Na+/Ca2+ exchanger current (INCX) triggered by spontaneous Ca2+ release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms (“Ca2+ clock model”). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between −40 and 40 mV (−80 ± 10.6 pA, at −20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca2+]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca2+]e, L-type Ca2+ channel (VOCCL) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K+ channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca2+ release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca2+ releases via RyR induce sharp increases in subsarcolemmal [Ca2+]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative “Ca2+ clock” would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.

Spontaneous inward currents reflecting oscillatory activation of Na+/Ca2+ exchangers in human embryonic stem cell-derived cardiomyocytes

Na(+)/Ca(2+) exchanger current (INCX) triggered by spontaneous Ca(2+) release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca(2+) clock model"). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between -40 and 40 mV (-80 ± 10.6 pA, at -20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca(2+)]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca(2+)]e, L-type Ca(2+) channel (VOCCL) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K(+) channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca(2+) release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca(2+) releases via RyR induce sharp increases in subsarcolemmal [Ca(2+)]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca(2+) clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.

Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~18weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

Super-Resolution Analysis of the Distribution of RyR, Cav1.2 and NCX within the Mammalian Couplon

We have developed a dynamically stable 3D dSTORM system, modified to give highly accurate blink positioning deep within a thick sample, to investigate the distribution and localization of the ryanodine receptor (RyR), the L-type calcium channel (Cav1.2) and the sodium-calcium exchanger (NCX) both on the surface and in the interior of the rat cardiomyocyte. The images have a resolution of 10nm in X and Y and 40 nm in Z, and cover areas of up to 1000 µm2 in XY with depths of up to 700nm in Z.We have been able to identify the position of individual RyR tetramers and have confirmed the recently published tomographic finding that in the resting state most RyR clusters are neither homogenous nor well-ordered. In addition, we found that surface RyR clusters are smaller both in extent and membership than those in the cell interior.Cav1.2 was commonly found in small spherical clusters of between 30-100 nm in diameter and were smaller and far denser than their RyR counterparts, with their centers appearing to be tightly packed. Cav1.2 clusters on the surface of the cell seemed little different from those in the interior, although the surface density was much higher. Super-clusters, made of 3 or more individual clusters were observed throughout the cell.NCX was more widely distributed and than either RyR or Cav1.2 and often formed a dense carpet on the cell surface made up of many small spherical clusters (∼50 nm dia). The mouth of the t-tubules were often very heavily labelled for 2-3 µm into the cell.

Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca2+ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.

Slow Ca2 + sparks de-synchronize Ca2 + release in failing cardiomyocytes: Evidence for altered configuration of Ca2 + release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca2+ transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca2+ release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca2+ transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152±12% of SHAM, P<0.05). The rise time of Ca2+ sparks was also increased in CHF (SHAM=9.6±0.6ms, CHF=13.2±0.7ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca2+ release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca2+ release across the cell. Dyssynchronous Ca2+ transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca2+ release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca2+ transients in CHF result from alterations in Ca2+ sparks, consistent with rearrangement of ryanodine receptors within Ca2+ release units. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

Electrical Remodeling in Dyssynchrony and Resynchronization

Heart failure (HF) is associated with anatomic and functional remodeling of cardiac tissues in both animal models and humans, which alters Ca(2+) homeostasis, protein phosphorylation, excitation-contraction coupling, results in arrhythmias. Indeed, the electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. The changes in cellular and tissue function are regionally heterogenous particularly in the dyssynchronously contracting heart. Cardiac resynchronization therapy (CRT) is widely applied in patients with HF and dyssynchronous left ventricular (LV) contraction (DHF), but the electrophysiological consequences of CRT are not fully understood. We demonstrated the molecular and cellular basis of excitability, conduction, and electrical remodeling in DHF and its restoration by CRT using a canine tachypacing HF model. CRT partially reversed the DHF-induced downregulation of K(+) current and improved Na(+) channel gating and abbreviated persistent (late) Na(+) current. CRT reduced Ca(2+)/calmodulin protein kinase II activity and restored transverse tubular system and spatial distribution of ryanodine receptor, thus it significantly improved Ca(2+) homeostasis especially in myocytes from late-activated, lateral wall and restored the DHF-induced blunted β-adrenergic receptor responsiveness. CRT abbreviated DHF-induced prolongation of APD in the lateral wall myocytes and reduced the LV regional gradient of APD and suppressed the development of early afterdepolarizations. In conclusion, CRT partially restores the DHF-induced ion channel remodeling, abnormal Ca(2+) homeostasis, blunted β-adrenergic response, and regional heterogeneity of APD, thus it may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improve mechanical performance of the heart.

Cardiac Excitation-Contraction Coupling Proteins: A 3D Spatial Analysis

The vital heart beat that pumps blood around the body is caused by millions of cardiac muscle cells exciting and contracting in concert. Cellular contraction is inextricably linked to the sarcoplasmic reticulum (SR) release event and subsequent binding of Ca2+ to myofibril binding sites. An integral protein in the release event is the SR membrane bound Ryanodine Receptor (RyR). Ca2+ enters the narrow spaces between transverse tubules and the termini of the SR via voltage-gated L-type Ca2+ channels. It then binds to RyR's to evoke the release of more Ca2+ from the SR, into the cytosol. Mathematical models examining this mechanism of excitation-contraction coupling (ECC) have thus far lacked anatomically realistic parameters for representing the ultrastructure and the distributions of key proteins in these myocytes. Our work has focused on producing an anatomically correct 3D model of a healthy cardiomyocyte that represents the spatial organization of the different cellular components involved in ECC. To this end, this study seeks to develop and perform statistical analyses on the 3D spatial distribution of RyR's and other ECC proteins in the healthy cell. Using immunofluorescence confocal microscopy images of rat ventricular muscle, we have carried out 3D spatial analyses which have enabled us to investigate the anisotropy, clustering, randomness and scatter of RyR's. We have also examined aspects of the spatial relationship between RyR's and myofibrils at Z-disks via the implementation of computational models. We have thus developed a novel anatomically based model of cardiomyocyte RyR distribution. This combined with models of other ECC component proteins, will enable us to gain new insights into the role of cardiomyocyte ultrastructure in determining the spatiotemporal changes in intracellular Ca2+ which is central in ECC.

Analysis of Ca v1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

We analyzed the distribution of ryanodine receptor (RyR) and Ca v1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Ca v1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Ca v1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Ca v1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Ca v1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Ca v1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Ca v1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Ca v1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca 2+ regulation and modeling Ca 2+ dynamics.

Spatiotemporal Profiles of Sarcoplasmic Reticulum Ca2+ Release in Mouse Atrial Cardiomyoctes in situ

Previous studies in atrial cardiomyocytes isolated from various mammalian species have demonstrated poorly developed transverse(t)-tubules and inhomogeneities of sarcoplasmic reticulum (SR) Ca2+ release, but little is known of mice. Here, we examined the t-tubular organization and action potential-evoked Ca2+ release in in situ myocytes, using confocal microscopy of Langendorff-perfused hearts. Imaging of ANNINE-6plus-stained sarcolemmal membranes revealed paucity of t-tubules in atrial, but densely and regularly spaced t-tubules in ventricular myocytes. However, both myocyte types exhibited regular, striated appearance of type 2 ryanodine receptor distribution. Line-scans (2 ms/line) across multiple myocytes obtained during sinus rhythm from fluo-4 loaded hearts revealed homogeneous [Ca2+]i increases in ventricular myocytes, whereas atrial myocytes exhibited areas with delayed transients (see Figure). Histograms of F50values (the time to 50% of peak F/F0 [where F indicates fluorescence intensity, and F0 indicates F at rest]) for the subsarcolemmal (SS) and central (C) compartments in ventricular myocytes were largely congruent, whereas the corresponding atrial histograms did not superimpose and exhibited multiple peaks. Thus, major myocyte-to-myocyte differences in the spatial organization of SR Ca2+ release exist among in situ mouse atrial myocytes, likely reflecting non-uniform t-tubule distribution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Imaging of the Ryanodine Receptor Distribution in Rat Cardiac Myocytes with Optical Single Channel Resolution

We have applied a new optical super-resolution technique based on single molecule localisation to examine the peripheral distribution of a cardiac signalling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. Using high-resolution antibody labeling data we show that the new imaging approach, termed localization microscopy, can give novel insight into the distribution of large proteins, with optical single channel resolution. We present, to our knowledge, the first direct data showing evidence for a two-dimensional array-like arrangement of RyRs in cardiac muscle. Morphological analysis of peripheral RyR clusters in the surface membrane revealed a mean size of ∼14 RyRs per cluster, almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often less than 50 nm suggesting that peripheral RyR clusters may exhibit strong inter-cluster signalling. The cluster size varied widely and followed a near-exponential distribution. We show that this distribution is compatible with a stochastic cluster assembly process and construct simple cluster growth models that generate size distributions very similar to our experimental observations. Based on the placement and morphology of RyR clusters we suggest that calcium sparks may be the result of the concerted activation of several clusters forming a functional ‘supercluster’ whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels. The new imaging approach can be extended to other cardiac proteins and should yield novel insight into excitation-contraction coupling and the control of cardiac contractility.