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

We present what we believe to be a new mathematical model of Ca(2+) leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca(2+)-release units, each containing a cluster of stochastically gating Ca(2+) channels (RyRs), whose biophysical properties (e.g., Ca(2+) sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca(2+)-release properties, and shows how this balances reuptake by the SR Ca(2+) pump. Simulations reveal that SR Ca(2+) leak consists of brief but frequent single RyR openings (~3000 cell(-1) s(-1)) that are likely to be experimentally undetectable, and are, therefore, "invisible". We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca(2+)), and occasionally lead to the generation of Ca(2+) sparks (~130 cell(-1) s(-1)). Furthermore, this physiological formulation of "invisible" leak allows for the removal of the ad hoc, non-RyR mediated Ca(2+) leak terms present in prior models. Finally, our model shows how Ca(2+) sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.

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Cardiac ryanodine receptor (RyR2) Ca 2þ release channels occur as multi-

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.

Precise positioning of the mitotic spindle is important for specifying the plane of cell division and the subsequent partitioning of the cell's contents to the daughter cells. Studies on different organisms and cell types have suggested diverse centering mechanisms: astral microtubules grow out from the spindle and push against the cortex, cortical dynein motors pull on astral microtubules, and dynein-dependent organelle transport on astral microtubules leads to a reactive force on the spindle. The different mechanisms lead to different predictions for the precision of centering, how mutations effect the precision, and the magnitude of the forces associated with spindle centering. We used image processing to accurately track the position and orientation of the mitotic spindle during the first cell division in the C. elegans embryo. The high precision of centering, < 1% of cell diameter transverse to the anterior-posterior axis, increased after RNAi against gpr-1/2, genes encoding activators of the cortical force generators; this suggests that centering is not mediated by gpr-1/2-dependent cortical pulling forces. To measure the forces associated with spindle positioning, we built a magnetic tweezers apparatus so that forces could be exerted on the spindle via beads incorporated into the embryo: forces of approximately 20 pN were required to displace the spindle through 1 mm. These mechanical experiments constrain molecular models of the centering process.

Calcium (Ca) sparks are elementary events of intracellular Ca signaling, which tend to occur randomly. Ca waves and whole-cell Ca oscillations occur under Ca overload and disease conditions. How Ca waves emerge from Ca sparks is not completely understood. We developed a three-dimensional model for Ca cycling which contains 100x20x10=20,000 identical Ca release units (CRUs), simulating the CRU network corresponding to a complete cardiac myocyte with dimensions of 100x20x10 micrometers. Using this model, we can generate the well known Ca signaling hierarchy: Ca quarks, Ca sparks, macro-sparks, abortive waves, and full Ca waves. We can also induce spiral waves within the cell, a wave phenomenon widely observed in myocyte experiments. Besides the well known experimental observation that increasing Ca loading promotes these wave dynamics, we also make the following observations: 1) The diffusion rate of Ca is a key parameter. Spontaneous Ca waves occur only when the diffusion rate is above a critical value. 2) When the model is homogeneous, Ca waves originate from different locations via a selforganizing process. This self-organizing process is influenced by, but does not require, heterogeneity. 3) When the model contains heterogeneities, such as heterogeneous Ca release channel distribution, Ca waves can originate from different locations or occur repeatedly from the same location. In real cardiac rabbit ventricular myocytes loaded with Fluo-4 AM to image intracellular Ca, Ca waves typically originate from different locations after successive rapid pacing episodes. In conclusion, our results indicate that Ca waves in cardiac myocytes originate predominantly as a result of self-organizing processes rather than pre-existing heterogeneities. 3026-Pos Board B131Ca 2D Leak and Ca 2D Sparks in Mammalian Heart: Insights from a Computational Model Calcium (Ca2þ) 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 Ca2þ 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 Ca2þ 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 Ca2þ entry via voltage gated L-type Ca2þ channels. The synchronized opening of the RyR2 cluster leads to localized elevations of...

Intracellular calcium handling is a complex nonlinear process that regulates cardiac contraction and rhythm, and cardiac arrhythmias have been associated with alterations in the calcium homeostasis. The spatiotemporal behavior of anomalous calcium handling includes a rich variety of local and global spontaneous release events ranging form isolated calcium sparks to multiple calcium waves originating at the same time in different parts of a single cardiomyocyte. We have developed a novel method for automatic detection and characterization of spontaneous calcium release events from a sequence of fluorescence microscopy images. First, a mask of the cell's shape is defined using a thresholding method in order to eliminate the effects of experimental background noise. A total fluorescence signal is then obtained by averaging pixel values within the mask for each frame, which is normalized to baseline fluorescence measured in frames without activity. The resulting signal presents a non-stationary behavior revealing the occurrence of different types of events. A wavelet-based detection method is used in order to identify independent global events in the average signal. Each event is then classified into one of three predefined types: a) single wave propagating across the cell, b) multiple simultaneous local events or c) a train of propagating waves (calcium bursts). Since the average signal does not contain local information it cannot be used to fully distinguish between these three event types. Therefore, we developed a classification method that uses a motion-tracking algorithm to identify each of the local release events contributing to a particular global event. This approach allows determining the trajectory, size, and propagation velocity of each local event and provides a reliable classification of global events. Furthermore, the method quantifies the contribution of each event type to the total calcium leakage during an experiment.