Calcium Release Units Research Articles

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age.

Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2(R33Q/R33Q) (R33Q) mutation. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2 wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial.

Shotgun proteomic analysis of sarcoplasmic reticulum preparations from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in excitation-contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty-three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation-contraction coupling and excitation-metabolism coupling.

Growth associated protein 43 is expressed in skeletal muscle fibers and is localized in proximity of mitochondria and calcium release units.

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.

Matters of the heart in bioenergetics: mitochondrial fusion into continuous reticulum is not needed for maximal respiratory activity

Mitochondria are dynamic structures for which fusion and fission are well characterized for rapidly dividing cells in culture. Based on these data, it has recently been proposed that high respiratory activity is the result of fusion and formation of mitochondrial reticulum, while fission results in fragmented mitochondria with low respiratory activity. In this work we test the validity of this new hypothesis by analyzing our own experimental data obtained in studies of isolated heart mitochondria, permeabilized cells of cardiac phenotype with different mitochondrial arrangement and dynamics. Additionally, we reviewed published data including electron tomographic investigation of mitochondrial membrane-associated structures in heart cells. Oxygraphic studies show that maximal ADP-dependent respiration rates are equally high both in isolated heart mitochondria and in permeabilized cardiomyocytes. On the contrary, these rates are three times lower in NB HL-1 cells with fused mitochondrial reticulum. Confocal and electron tomographic studies show that there is no mitochondrial reticulum in cardiac cells, known to contain 5,000-10,000 individual, single mitochondria, which are regularly arranged at the level of sarcomeres and are at Z-lines separated from each other by membrane structures, including the T-tubular system in close connection to the sarcoplasmic reticulum. The new structural data in the literature show a principal role for the elaborated T-tubular system in organization of cell metabolism by supplying calcium, oxygen and substrates from the extracellular medium into local domains of the cardiac cells for calcium cycling within Calcium Release Units, associated with respiration and its regulation in Intracellular Energetic Units.

Modelling cardiac calcium sparks in a three‐dimensional reconstruction of a calcium release unit

Triggered release of Ca2+ from an individual sarcoplasmic reticulum (SR) Ca(2+) release unit (CRU) is the fundamental event of cardiac excitation–contraction coupling, and spontaneous release events (sparks) are the major contributor to diastolic Ca(2+) leak in cardiomyocytes. Previous model studies have predicted that the duration and magnitude of the spark is determined by the local CRU geometry, as well as the localization and density of Ca(2+) handling proteins. We have created a detailed computational model of a CRU, and developed novel tools to generate the computational geometry from electron tomographic images. Ca(2+) diffusion was modelled within the SR and the cytosol to examine the effects of localization and density of the Na(+)/Ca(2+) exchanger, sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA), and calsequestrin on spark dynamics. We reconcile previous model predictions of approximately 90% local Ca(2+) depletion in junctional SR, with experimental reports of about 40%. This analysis supports the hypothesis that dye kinetics and optical averaging effects can have a significant impact on measures of spark dynamics. Our model also predicts that distributing calsequestrin within non-junctional Z-disc SR compartments, in addition to the junctional compartment, prolongs spark release time as reported by Fluo5. By pumping Ca(2+) back into the SR during a release, SERCA is able to prolong a Ca(2+) spark, and this may contribute to SERCA-dependent changes in Ca(2+) wave speed. Finally, we show that including the Na(+)/Ca(2+) exchanger inside the dyadic cleft does not alter local [Ca(2+)] during a spark.

Calcium release units in heart failure: that's about the size of it

This editorial refers to ‘Ultrastructural remodelling of Ca2+ signalling apparatus in failing heart cells’ by H.-D. Wu et al ., pp. 430–438, this issue. Cardiac remodelling, often defined as changes in the mass and shape of the whole heart,1 has been a focus of research over several decades. Despite considerable efforts, the cellular and molecular mechanisms that lead to contractile dysfunction of the remodelled heart are still not clear. Remodelling may also occur at the level of the cardiomyocyte and is then termed ultrastructural remodelling. Studies have revealed substantial ultrastructural alterations in several parts of the cardiomyocytes during cardiac disease, including the sarcoplasmic reticulum (SR)2 and transverse (T)-tubules,3,4 and in the positioning of ion channels and pumps that are crucial in regulating myocardial contraction and relaxation. Thus, it is likely that ultrastructural remodelling can explain several important aspects of systolic and diastolic heart failure of various aetiologies. The machinery that couples the electrical activation of the cardiomyocyte to mechanical contraction is set to control transient rises of [Ca2+] in the cytosol. Research in the past decades has revealed that the global rise of [Ca2+] in the cytosol that activates myofilaments is regulated by mechanisms that are confined within spatially constrained microdomains in the cardiomyocyte. These microdomains occur at sites where Ca2+ channels in the T-tubule membrane come very close to Ca …

Ultrastructure of cardiac muscle in reptiles and birds: optimizing and/or reducing the probability of transmission between calcium release units

It is known that cardiac myocytes contain three categories of calcium release units (CRUs) all bearing arrays of RyR2: peripheral couplings, constituted of an association of the junctional SR (jSR) with the plasmalemma; dyads, associations between jSR and T tubules; internal extended junctional jSR (EjSR)/corbular jSR that is not associated with plasmalemma/T tubules. The bird hearts, even if fast beating (e.g., in finch and hummingbird) have no T tubules, despite fiber sizes comparable to those of mammalian ventricle, but are rich in EjSR/corbular SR. The heart of small lizard also lacks T tubule, but it has only peripheral couplings and compensates for lack of internal CRUs by the small diameter of its cells. We have extended previous information on chicken heart to finch and lizard by establishing a spatial relationship between RyR2 clusters in jSR of peripheral couplings and clusters of intra-membrane particles identifiable as voltage sensitive calcium channels (CaV1.2) in the adjacent plasmalemma. This provides the structural basis for initiation of the heart beat in all three species. Further we evaluated the distances separating peripheral couplings from each other and between EjSR/corbular SR sites within the bird muscles in all three hearts. The distances suggest that peripheral coupling sites are most likely to act independently of each other and that a calcium wave-front propagation from one internal CRU site to the other across the level of the Z line, may be marginally successful in the chicken, but certainly very effective in the finch.

Ryanodine Receptor Recruitment and Construction of Calcium Release Sites in Cardiac Myocytes

Local character of calcium release in cardiac myocytes implies independent recruitment of calcium release units by triggering stimuli. Calcium release from individual CRUs also displays quantal character, interpreted either as recruitment of small cohorts of independent RYRs (1,2), or as recruitment of small cohorts of RYR clusters with coupled RYRs gating (3). We tested both interpretations on published experimental data using a model of virtual calcium release units (vCRUs) consisting of 1-10 clusters that were constructed in accordance with the experimentally observed cluster size distribution (4). If RYR gating was independent, vCRUs consisting of 3 or more clusters provided a good agreement between the model and experimental data (1). If RYR gating was coupled, the calcium release flux of vCRUs did not display quantal structure that would correspond to in situ observations. The model of RYR gating (2) combined with the model of independent RYRs in vCRUs (2) matched the experimentally observed calcium dependence of calcium spark frequency (5) under all conditions. However, the Mg2+-binding parameters of RYRs were in accordance with the single-channel observations (6) only for vCRUs composed of 3 or more clusters. In conclusion, these results favor independent over fully coupled RYR gating in situ, and predict the presence of at least 40 RYRs per cardiac calcium release unit. Supported by grants APVV-0441-09, APVV-0721-10, VEGA 2/0190/10, 2/0203/11, and 2/0197/11.

Stochastic Simulation Assessing the Functional Changes Occurring during Heart Failure

Heart failure has a high prevalence in the general population and mortality rates that remain excessive. Heart failure is characterized by a the loss of the ability of the heart to pump blood and an increased propensity for fatal arrhythmias. Changes in excitation-contraction coupling occur in heart failure and involve the down regulation of sarcolemmal potassium channels, increased expression of the sodium-calcium exchanger, decreased SR calcium ATPase expression, as well are remodeling of the T-tubule network. These changes are aggravated by chronically increased beta-adrenergic stimulation which has been characterized as a maladaptive mechanism. In HF, a chronic hyperadrenerigic state results in chronic hyperphosphorlyation of the cardiac ryanodine receptor leading to increased sensitivity for calcium dependent channel activation. We have developed a computational model of excitation-contraction coupling (that improves upon Williams et al., 2011) based on the rat ventricular myocyte that includes the 20,000 calcium release units, each containing a cluster of 49 ryanodine receptors and 7 L-type calcium channels both of which gate stochastically. The model also includes the membrane currents that govern the action potential and the remaining components of calcium dynamics. The model is solved using our Ultra-fast Monte Carlo Algorithm and GPGPUs that enable this massive calculation to be solved efficiently. By implement changes in ion channel and transporter behavior in the model while also including the spatial re-organization of the TT system, we are able to examine how action potentials and Ca2+ signaling changes in HF. This work reveals how cellular Ca2+ instability arises and how late diastolic calcium release events (i.e. Ca2+ sparks) and increased diastolic leak underlie the propensity for development of arrhythmogenic calcium waves at the cellular level.

Refractoriness of Calcium Release Units in Rat Cardiac Myocytes

Calcium release units (CRUs) in cardiac myocytes contain a large number of ryanodine receptors (RyRs), but it seems that only a small fraction of RyRs is being activated during a single calcium release event. Repetitive activity of CRUs is not well understood from the point of refractoriness. We used direct measurement of Ca-spikes activated by calcium current (ICa) to unveil the refractoriness of CRU firing from the properties and occurrence of calcium spikes under control conditions and in the presence of the DHPR activator FPL.ICa was activated by voltage pulses from 50 to 0 mV under whole-cell patch-clamp. Ca-spikes were measured by confocal microscopy using Ca2+ indicators (Fluo-3 and Oregon Green BAPTA-5N) in the presence of EGTA. A large majority of CRUs responded to stimulation by a single Ca-spike, while 13.4 % produced two subsequent (twin) Ca-spikes. Early Ca-spikes (single spikes and the first of twin Ca-spikes) had similar latencies, while the second Ca-spikes were significantly delayed. Amplitude distribution of the early Ca-spikes consisted of four quantal levels with equal amplitudes and binomially distributed frequency of occurrence, supporting the hypothesis that different Ca-spike amplitudes are due to a different number of independent quantal levels, likely individual RYR openings. The probability of occurrence of second Ca-spikes was inversely proportional to the quantal size of the early Ca-spike. In the presence of FPL, the probability of occurrence of twin spikes was decreased relative to control, despite persistent activation of DHPR receptors.We conclude that refractoriness of local calcium release does not result from Ca-dependent DHPR inactivation, but rather it is caused by inactivation of the release unit due to local depletion of the SR.Supported by APVV-0721-10, APVV-0441-09, VEGA 2/0203/11, VEGA 2/0917/11, and VEGA 2/0190/10.

Construction of Calcium Release Sites in Cardiac Myocytes

Local character of calcium release in cardiac myocytes, as defined by confocal recordings of calcium sparks, implies independent activation of individual calcium release sites based on ryanodine receptor (RyR) channel recruitment. We constructed virtual calcium release sites (vCRSs) composed of a variable number of RyR channels distributed in clusters in accordance with the experimentally observed cluster size distribution. The vCRSs consisted either of a single virtual calcium release unit (vCRU), in which all clusters shared a common dyadic space, or of multiple virtual calcium release units (CRUs) containing one cluster each and having separate dyadic spaces. We explored the stochastic behavior of vCRSs to understand the activation and recruitment of RyRs during calcium sparks. RyRs were represented by the published allosteric gating model that included regulation by cytosolic Ca2+ and Mg2+. The interaction of Mg2+ with the RyR Ca2+-binding sites and the refractory period of vCRSs were optimized to accord with the experimentally observed calcium dependence of calcium spark frequency. The Mg2+-binding parameters of RyRs that provided the best description of spark frequency depended on the number of RyRs assembled in the vCRSs. Adequate inhibitory effect of Mg2+ on the calcium dependence of RyR open probability was achieved if the vCRSs contained at least three clusters. For the distribution of the number of open RyRs in evoked calcium sparks to correspond to the experimentally observed distribution of spark calcium release fluxes, at least three clusters had to share a common virtual CRU, in which ∼3 RyRs open to form an average spark. These results reconcile the small cluster size and stochastic placement of RyRs in the release sites with the estimates of the amount of RyR protein, volume density of calcium release sites, and the size of calcium release sites in rat cardiac myocytes.

Transverse tubules are a common feature in large mammalian atrial myocytes including human

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 μm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.

Stochastic simulation of cardiac ventricular myocyte calcium dynamics and waves.

A three dimensional model of calcium dynamics in the rat ventricular myocyte was developed to study the mechanism of calcium homeostasis and pathological calcium dynamics during calcium overload. The model contains 20,000 calcium release units (CRUs) each containing 49 ryanodine receptors. The model simulates calcium sparks with a realistic spontaneous calcium spark rate. It suggests that in addition to the calcium spark-based leak, there is an invisible calcium leak caused by the stochastic opening of a small number of ryanodine receptors in each CRU without triggering a calcium spark. The model also explores the mechanism of calcium wave propagation between release sites under the conditions of calcium overload.

Ca 2+ Leak and Ca 2+ Sparks in Mammalian Heart: Insights from a Computational Model

Calcium (Ca2+) signaling in muscle, neuronal, and non-excitable cells has benefited significantly from advances in biological tools and imaging technology, however, the molecular interactions of nanoscopic molecules, structures and compartments has been challenging to study under physiological conditions. Here, we exploit novel computational modeling techniques to examine real-time molecular and cellular physiology in cardiac ventricular myocytes. The model focuses on local and cell-wide Ca2+ signaling phenomena related to calcium induced calcium release from intracellular calcium channels, ryanodine receptors (RyR2s), located on the sarcoplasmic reticulum (SR) membrane. This work is informed by the latest molecular investigations and recent characterizations of channels, transporters, and buffers located in mammalian heart. We have created a detailed, whole-cell model of Ca2+ signaling using a realistic number of calcium release units (CRU) each containing a cluster of stochastically gating RyR2s. During systole the opening of these RyR2s is triggered by Ca2+ entry via voltage gated L-type Ca2+ channels. The synchronized opening of the RyR2 cluster leads to localized elevations of [Ca2+]i known as Ca2+ sparks. During diastole Ca2+ sparks are still observed and are attributed to the finite opening rate of the RyR2. RyR2s are also believed to display unsynchronized or non-spark openings where only a few channels in the CRU open without triggering the remainder of the RyR2 cluster. This non-spark Ca2+ release would be below current experimental detection thresholds and therefore “invisible.” These spark and non-spark openings of RyR2s constitute a molecular basis for Ca2+ leak from the SR. The computational model suggests that a significant fraction of SR Ca2+ leak is due to RyR2s openings that fail to trigger a “visible” Ca2+ spark. Additionally, the fraction of non-spark or “invisible” SR Ca2+ leak increases as SR Ca2+ content declines.

The evolution of the mitochondria-to-calcium release units relationship in vertebrate skeletal muscles.

The spatial relationship between mitochondria and the membrane systems, more specifically the calcium release units (CRUs) of skeletal muscle, is of profound functional significance. CRUs are the sites at which Ca2+ is released from the sarcoplasmic reticulum during muscle activation. Close mitochondrion-CRU proximity allows the organelles to take up Ca2+ and thus stimulate aerobic metabolism. Skeletal muscles of most mammals display an extensive, developmentally regulated, close mitochondrion-CRU association, fostered by tethering links between the organelles. A comparative look at the vertebrate subphylum however shows that this specific association is only present in the higher vertebrates (mammals). Muscles in all other vertebrates, even if capable of fast activity, rely on a less precise and more limited mitochondrion-CRU proximity, despite some tethering connections. This is most evident in fish muscles. Clustering of free subsarcolemmal mitochondria in proximity of capillaries is also more frequently achieved in mammalian than in other vertebrates.

Structural evidence for perinuclear calcium microdomains in cardiac myocytes

At each heartbeat, cardiac myocytes are activated by a cytoplasmic Ca2+ transient in great part due to Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs) clustered within calcium release units (peripheral couplings/dyads). A Ca2+ transient also occurs in the nucleoplasm, following the cytoplasmic transient with some delay. Under conditions where the InsP3 production is stimulated, these Ca2+ transients are regulated actively, presumably by an additional release of Ca2+ via InsP3 receptors (InsP3Rs). This raises the question whether InsP3Rs are appropriately located for this effect and whether sources of InsP3 and Ca2+ are available for their activation. We have defined the structural basis for InsP3R activity at the nucleus, using immunolabeling for confocal microscopy and freeze-drying/shadowing, T tubule “staining” and thin sectioning for electron microscopy. By these means we establish the presence of InsP3R at the outer nuclear envelope and show a close spatial relationship between the nuclear envelope, T tubules (a likely source of InsP3) and dyads (the known source of Ca2+). The frequency, distribution and distance from the nucleus of T tubules and dyads appropriately establish local perinuclear Ca2+ microdomains in cardiac myocytes.

Frequency and release flux of calcium sparks in rat cardiac myocytes: a relation to RYR gating

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca2+ releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca2+ and Mg2+ ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg2+. Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg4RYR and CaMg3RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca2Mg2RYR and Ca3MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg2+ unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.

Calsequestrin mediates changes in spontaneous calcium release profiles

Calsequestrin (CSQ) is the primary calcium buffer within the sarcoplasmic reticulum (SR) of cardiac cells. It has also been identified as a regulator of Ryanodine receptor (RyR) calcium release channels by serving as a SR luminal sensor. When calsequestrin is free and unbound to calcium, it can bind to RyR and desensitize the channel from cytoplasmic calcium activation. In this paper, we study the role of CSQ as a buffer and RyR luminal sensor using a mechanistic model of RyR–CSQ interaction. By using various asymptotic approximations and mean first exit time calculation, we derive a minimal model of a calcium release unit which includes CSQ dependence. Using this model, we then analyze the effect of changing CSQ expression on the calcium release profile and the rate of spontaneous calcium release. We show that because of its buffering capability, increasing CSQ increases the spark duration and size. However, because of luminal sensing effects, increasing CSQ depresses the basal spark rate and increases the critical SR level for calcium release termination. Finally, we show that with increased bulk cytoplasmic calcium concentration, the CRU model exhibits deterministic oscillations.

Progressive Triad-Mitochondria Un-Coupling in Aging

An impairment of the mechanisms controlling the release of calcium from internal stores (excitation-contraction (EC) coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging (EC un-coupling theory). EC coupling in muscle fibers occurs at specialized intracellular junctions called calcium release units, or triads, which are specifically placed at sarcomere's I-A band transition. In recent publications we have shown that: a) in human muscle, the frequency of triads decreases significantly with age (Boncompagni et al., 2006; J Gerontol 61:995); and b) in mice, triads are tethered to mitochondria placed at the I band (Boncompagni et al., 2009; MBC 20:1059). Here we have studied the frequency, sarcomeric-localization, ultrastructure, and coupling of triads/mitochondria in EDL from male WT mice using transmission electron microscopy (TEM).Preliminary results indicates that the number of triads/100μm2 of longitudinal section in aging mice (n=4, 25-35 months of age) decreases compared to the adult mice (n=5, 3-12 months of age): 92±9 vs. 79±8. In addition, the percentage of abnormally positioned triads (longitudinal and/or oblique) increases. On the other hand, the total volume of mitochondria does not change significantly with age. However, the number of mitochondria-profiles/100μm2 of longitudinal section is reduced (54±7 vs. 43±6), suggesting a remodelling/fusion of these organelles. Finally, we have assessed the positioning of mitochondria in respect to myofibrils and triads: a) the number of mitochondria at the A band (misplaced) slightly increases with age (9% vs 3%), whereas the number of triads-mitochondria couples is significantly reduced: 39±5 vs. 26±4. Our observations indicates: a) a age-related partial disarrangement and spatial re-organization of EC coupling/mitochondrial apparatuses; and b) a decreased percentage of mitochondria functionally tethered to calcium release sites. This could in part explain the decline of muscle performance associated to increasing age.