Dyadic Cleft Research Articles

Stochastic Binding of Ca 2+ Ions in the Dyadic Cleft; Continuous versus Random Walk Description of Diffusion

Ca 2+ signaling in the dyadic cleft in ventricular myocytes is fundamentally discrete and stochastic. We study the stochastic binding of single Ca 2+ ions to receptors in the cleft using two different models of diffusion: a stochastic and discrete Random Walk (RW) model, and a deterministic continuous model. We investigate whether the latter model, together with a stochastic receptor model, can reproduce binding events registered in fully stochastic RW simulations. By evaluating the continuous model goodness-of-fit for a large range of parameters, we present evidence that it can. Further, we show that the large fluctuations in binding rate observed at the level of single time-steps are integrated and smoothed at the larger timescale of binding events, which explains the continuous model goodness-of-fit. With these results we demonstrate that the stochasticity and discreteness of the Ca 2+ signaling in the dyadic cleft, determined by single binding events, can be described using a deterministic model of Ca 2+ diffusion together with a stochastic model of the binding events, for a specific range of physiological relevant parameters. Time-consuming RW simulations can thus be avoided. We also present a new analytical model of bimolecular binding probabilities, which we use in the RW simulations and the statistical analysis.

Excitation-Contraction Coupling - II

2801-Pos Board B571 Increased miR-129 in Heart Failure Leads to Diminished Calcium Release by Downregulation of Junctophilin 4 Jennifer A. Rochira1, Karim Roder1, Weiyan Li1, Radmila Terentyeva2, Yukiko Kunitomo3, Terry S. Elton4, Dmitry Terentyev1. RI Hospital/Brown University, Providence, RI, USA, RI Hospital, Providence, RI, USA, Brown University, Providence, RI, USA, Ohio State University, Columbus, OH, USA. MicroRNAs (miRNAs) are small nonprotein-coding RNA molecules that target mRNAs for translational repression or degradation. Recent studies demonstrated that miRNA expression levels are substantially altered in heart failure (HF) and therefore may play a role in the functional abnormalities observed in diseased hearts. Since miR-129 expression is augmented in HF, we hypothesized that HF-observed changes in Excitation-Contraction Coupling result, in part, from miR-129-mediated downregulation of specific calcium handling proteins. Confocal Ca2þ imaging revealed that in electrically stimulated myocytes overexpressing miR-129, Ca2þ transient amplitudes were significantly reduced compared to controls. In contrast, sarcoplasmic reticulum (SR) Ca2þ contents assessed by rapid application of caffeine remained unchanged. Furthermore, kinetic analyses of Ca2þ transient decays suggested miR-129 overexpression does not affect Naþ/Ca2þ exchanger or SR Ca2þ ATPase function. Voltage-clamp experiments showed no difference in Ca2þ currents in miR-129 overexpressing and control cells. Additionally, permeabilized myocytes demonstrated no change in Ca2þ spark frequency nor amplitude indicating no effect from miR-129 overexpression on SR Ca2þ release channel Ryanodine Receptor (RyR2) function. Detailed analysis of Ca2þ transient rising phase revealed significant time-to-peak prolongation accompanied by reduction in Ca2þ release synchronization in miR-129 overexpressing myocytes compared with controls. Overall, these data imply that miR-129 overexpression reduces the fidelity of coupling between plasmalemmal Ca2þ channels and RyR2s located in junctional SR. Efficient coupling between L-type Ca2þ channels and RyR2 is facilitated by junctophilins (JPH), proteins tethering T-tubules to SR forming junctional membrane complexes keeping channels at precise distances. Western blot analyses demonstrated that miR-129 overexpression significantly reduced levels of JPH4, a putative target of miR-129. Therefore, our results suggest that increased levels of miR-129 may underlie diminished Ca2þ release in HF by downregulation of JPH4 thus altering geometry of the dyadic cleft.

Stochastic binding of Ca2+ ions in the dyadic cleft; continuous vs random walk description of diffusion

AbstractCa2+ signaling in the dyadic cleft in ventricular myocytes is fundamentally discrete and stochastic. In this paper we study the stochastic binding of single Ca2+ ions to receptors in the cleft using two different models of diffusion; a stochastic and discrete Random walk (RW) model, and a deterministic continuous model. We investigate if the latter model, together with a stochastic receptor model, can reproduce binding events registered in fully stochastic RW simulations. By evaluating the continuous model goodness‐of‐fit, we present evidences that it can. The large fluctuations in binding rate observed at the time level of single time steps are integrated and smoothed at the larger time scale of binding events, explaining the continuous model goodness‐of‐fit. With this we demonstrate that the stochasticity and discreteness of the Ca2+ signaling in the dyadic cleft, determined by single binding events, can be described with a deterministic model of Ca2+ diffusion together with a stochastic model of the binding events. Time consuming RW simulations can thus be avoided. We also present a new analytical model of bi‐molecular binding probabilities that is used in the RW simulations, and in the statistical analysis. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Abstract 491: Triggered Sarcoplasmic Reticulum Ca Release Mediated By Na-ca Exchanger And Brain Na Channels In Rabbit Ventricular Cells

We tested the hypothesis that sarcoplasmic reticulum (SR) Ca release triggered by cardiac action potentials is significantly affected by Na influx through brain isoforms of the Na channel. These channels are reportedly located in the t-tubules and could therefore activate reverse Na-Ca exchanger (NCX) and contribute to triggered release. In the complete absence of a Na gradient (Li replacement) Ca transients evoked by action potentials were delayed, their upstrokes slowed and their peak values diminished (SR Ca content not depleted under these conditions). Ca spikes recorded under these conditions occurred at significantly lower probability and became asynchronous (see figure ). Tetrodotoxin 200 nM (this concentration selectively blocks brain Na channel isoforms Nav 1.1, 1.3 and 1.6) reduced the magnitude and rate of rise of Ca transients in a way that resembled the effect of removing the Na gradient. We suggest that early activation of brain type Na channels leads to elevation of Na in the dyadic cleft. This causes reverse NCX, which primes the junction with Ca before L-type Ca channels (LCC) have opened. Since the junction has been primed with Ca, short LCC openings can effectively gate ryanodine receptors (RyRs). Because these LCC openings have short latencies and occur with high probability they produce spikes with short latencies at high probability. In the absence of a Na gradient longer LCC openings are required to fill the junction and gate RyRs. These occur with lower probability and greater latency. We conclude that brain Na channels together with NCX significantly increase LCC-RyR coupling fidelity and synchronize spikes.

The Cardiac Sarcoplasmic Reticulum

Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is the cornerstone of cardiac excitation-contraction coupling and Ca2+ signaling.1,2 However, as an amplification mechanism exhibiting a high degree of positive feed-back, it has to be kept in check by inhibitory systems to prevent spontaneous oscillatory Ca2+ releases which could possibly trigger cardiac arrhythmias. Local control theory provides us with an initial frame-work to understand how this could be accomplished.3 Mutually independent Ca2+ signaling events (Ca2+ sparks4) generate the necessary amplification locally without spreading to neighboring Ca2+ release sites, whereas the normal signal transduction from L-type Ca2+ channels to the SR occurs within the microdomain of the dyadic cleft, well isolated from the bulk of the cytosol. Uncoupling between neighboring Ca2+ spark sites thus ensures the reliability of the CICR system and occurs by virtue of steep concentration gradients away from the microdomain of Ca2+ release, and by means of the relative insensitivity of the SR Ca2+ release channels (ryanodine receptors [RyRs]) toward cytosolic Ca2+ triggers.5 This uncoupling by local control also underlies the observation that Ca2+ sparks occurring spontaneously remain localized and do not initiate a chain reaction of Ca2+ sparks traveling along the entire myocyte as a Ca2+ wave. However, this system can also become unstable and CICR is capable to override local control and to trigger oscillatory Ca2+ signals in cardiomyocytes, particularly under pathophysiological conditions. Waves of contractions traveling along isolated cardiomyocytes have been discovered and the underlying Ca2+ …

Spatial and temporal Ca 2+, Mg 2+, and ATP 2− dynamics in cardiac dyads during calcium release

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP 2− with Ca 2+ and Mg 2+ ions on spatiotemporal concentration profiles of Ca 2+, Mg 2+, and ATP 2− in the dyadic cleft during Ca 2+ release. The model revealed that Ca 2+ concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca 2+] in the dyadic space reached values similar to estimates of luminal [Ca 2+] in ∼1 ms, suggesting that during calcium release the Ca 2+ gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca 2+ bound to ATP 2−, and thus substantially decreased ATP 2− concentration in the dyadic space. The released Ca 2+ could also replace Mg 2+ in its complex with ATP 2− during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca 2+, Mg 2+, and ATP 2− might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.

Contribution of the Na +/Ca 2+ Exchanger to Rapid Ca 2+ Release in Cardiomyocytes

Trigger Ca 2+ is considered to be the Ca 2+ current through the L-type Ca 2+ channel (LTCC) that causes release of Ca 2+ from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current ( I Ca). In this article, we investigate the contribution of the Na +/Ca 2+ exchanger (NCX) to the trigger Ca 2+. Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na +/Ca 2+ exchange delays the Ca 2+ transient by 3–4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na + and Ca 2+ is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na + channel is present in the fuzzy space, the NCX is able to bring enough Ca 2+ into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na + channel and NCX.

A 3D Monte Carlo Analysis of the Role of Dyadic Space Geometry in Spark Generation

In multiple biological systems, vital intracellular signaling processes occur locally in minute periplasmic subspaces often referred to as signaling microdomains. The number of signaling molecules in these microdomains is small enough to render the notion of continuous concentration changes invalid, such that signaling events are better described using stochastic rather than deterministic methods. Of particular interest is the dyadic cleft in the cardiac myocyte, where short-lived, local increases in intracellular Ca 2+ known as Ca 2+ sparks regulate excitation-contraction coupling. The geometry of dyadic spaces can alter in disease and development and display significant interspecies variability. We created and studied a 3D Monte Carlo model of the dyadic cleft, specifying the spatial localization of L-type Ca 2+ channels and ryanodine receptors. Our analysis revealed how reaction specificity and efficiency are regulated by microdomain geometry as well as the physical separation of signaling molecules into functional complexes. The spark amplitude and rise time were found to be highly dependent on the concentration of activated channels per dyadic cleft and on the intermembrane separation, but not very sensitive to other cleft dimensions. The role of L-type Ca 2+ channel and ryanodine receptor phosphorylation was also examined. We anticipate that this modeling approach may be applied to other systems (e.g., neuronal growth cones and chemotactic cells) to create a general description of stochastic events in Ca 2+ signaling.

Quantification of Calcium Entry at the T-Tubules and Surface Membrane in Rat Ventricular Myocytes

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by ∼60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 μmol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 μmol/L cytosol, respectively). However, more Ca entry occurs per μm 2 of junctional membrane at the cell surface than in the T-tubules (in nM/ μm 2: 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions (∼35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.