RyR activity at resting condition. However, SR Ca leak in atrial myocytes can be facilitated significantly during activation of IP 3 -dependent signaling path- ways. 1411-Pos Board B255 Changes In Cytosolic Ca 2þ Have Greater Effects On SR Ca 2þ Leak Than Changes In SR Ca 2þ Frank J. Fabris, Onika Noel, Eric A. Sobie. Mount Sinai School of Medicine, New York, NY, USA. Attention has recently focused on preventing arrhythmias by controlling sarco- plasmic reticulum (SR) Ca 2þ ‘‘leak’’. Increased leak in ventricular myocytes is associated with regenerative Ca 2þ waves and delayed afterdepolarizations, leading to arrhythmias. Studies that have measured SR Ca 2þ leak have not examined changes in [Ca 2þ ] SR independent of changes in [Ca 2þ ] i , causing a de- gree of uncertainty as to which factor plays a greater role. Our current work ex- plores the possibility that changes in [Ca 2þ ] i have a greater effect on leak than changes in [Ca 2þ ] SR . In quiescent rat ventricular myocytes, we recorded steady-state Ca 2þ levels, then blocked the ryanodine receptors (RyRs) with a saturating concentration of tetracaine. Using the calcium indicator fluo-3, we recorded changes in [Ca 2þ ] i using a confocal microscope and analyzed the data using leak calcula- tions that took into account underlying assumptions about cytosolic and SR buffers. When extracellular Ca 2þ ([Ca 2þ ] e ) was increased from 0.5 mM to 1.0 mM at rest, leak increased 37% (951.019 vs. 12.351.108 mM/s), [Ca 2þ ] i increased 6.6% (98.9 50.09 vs. 105.453.2 nM, and [Ca 2þ ] SR decreased 5.2% (489521 vs. 464523 mM). We also compared leak in resting cells versus leak in the same cells immediately after pacing for 10 s at 1 Hz. At 1 mM [Ca 2þ ] e , pacing increased leak by 17.9% (12.351.108 vs. 14.558.8 mM/s), increased [Ca 2þ ] i by 9.4% (105.453.2 vs. 115.355.7 nM), but increased [Ca 2þ ] SR by only 1.0% (463.7522.7 vs. 468.2526.8 mM). Qualitatively similar results were obtained after pacing in 0.5 mmol [Ca 2þ ] e. These results suggest that [Ca] i plays a larger role in determining diastolic SR Ca 2þ leak than [Ca] SR . 1412-Pos Board B256 EPAC Does Not Affect Diastolic Sarcoplasmic Reticulum Ca 2þ Release in Rabbit Ventricular Myocytes Demetrio J. Santiago, Jerald W. Curran, Thomas R. Shannon. Rush University, Chicago, IL, USA. Recent evidence gathered in ventricular myocytes from rodents points out that the EPAC pathway is a strong promoter of the release of Ca 2þ from the sarco- plasmic reticulum (SR; Pereira et al., 2007, J. Physiol. 583:685-94). Encour- aged by the above observations, we studied the effects of 2 mM 8-CPT (a specific EPAC activator) on the diastolic SR Ca 2þ release in rabbit ventricular myocytes. Our initial studies used epifluorescence and focused on the relation- ship between the SR load and the diastolic SR Ca 2þ release (i.e., the so called SR Ca 2þ leak-load relationship). Contrary to the observations in rodents, our rabbit ventricular myocytes displayed no alterations of the leak-load relation- ship upon 8-CPT application. Since the leak-load relationship requires a steady state to be reached prior to the measurements, we also tested for non steady- state effects of EPAC stimulation using confocal microscopy. We studied the frequency and properties of Ca 2þ sparks during the first 30 seconds of rest de- cay following 2 minutes of 8-CPT application and field stimulation at 1 Hz. Our results showed no effects of EPAC stimulation on the spark frequency or the spatio-temporal properties of the sparks. In summary, our results suggest that EPAC does not affect diastolic SR Ca 2þ release in rabbit ventricular myocytes. Future studies will target the species dependence of the effect of EPAC and the effect of SR [Ca 2þ ] upon the release properties under these conditions. Calcium Fluxes, Sparks, and Waves II 1413-Pos Board B257 Ca Alternans in Cardiac Myocytes: Relating Macroscopic Behavior to Microscopic Ca Release Properties Robert J. Rovetti 1 , Alan Garfinkel 2 , James N. Weiss 3 , Zhilin Qu 3 . 1 Dept. of Mathematics, Loyola Marymount University, Los Angeles, CA, USA, 2 Depts. of Medicine (Cardiology) and Physiology, University of California Los Angeles, Los Angeles, CA, USA, 3 Dept. of Medicine (Cardiology), University of California Los Angeles, Los Angeles, CA, USA. Beat-to-beat alternation in the intracellular Ca (Ca i ) transient (Ca i alternans) causes pulsus alternans and electrocardiographic T-wave alternans, conditions associated with cardiac arrhythmias and sudden death. The whole cell Ca i tran- sient represents the summed activity of thousands of individual Ca sparks, i.e. Ca released locally by Ca release units (CRU). However, the extent to which the global behavior of the whole cell Ca i transient mirrors the microscopic behavior of individual CRU units is unclear. We derived a one-dimensional iterated map of CRU behavior in which we could independently adjust the probabilities of random triggering of Ca sparks, recruitment of Ca sparks from adjacent CRUs, and CRU refractoriness following a Ca spark. After ver- ifying that these three local properties (randomness, recruitment and refracto- riness) could sustain an ensemble (global) alternans in a two-dimensional cel- lular automata network, we developed a physiologically-detailed subcellular Ca cycling model containing a network of coupled stochastic CRU which rep- licated the iterated map predictions. We find that a number of experimentally- reported phenomena, including whole cell Ca i alternans, Ca waves in the pres- ence of high spatial cooperativity, graded whole-cell Ca release, and a steep de- pendence of fractional SR Ca release on SR Ca load, emerge naturally from the collective behavior of individual CRUs depending on the balance of these three properties. A striking prediction is that microscopic CRU behavior does not al- ways mirror collective CRU behavior, e.g. during whole cell Ca i alternans, Ca sparks from individual CRU do not consistently alternate. In addition, whole cell Ca i alternans is not generally dependent on alternans of diastolic SR Ca content. The findings provide novel multiscale insights into how global Ca signaling properties emerge from simple microscopic CRU properties. 1414-Pos Board B258 Analysis of Calcium Alternans in a Cardiac Myocyte Model that Uses Moment Equations to Represent Heterogenous Junctional SR Calcium Marco Huertas 1 , Gregory D. Smith 1 , Sandor Gyorke 2 . 1 College of William and Mary, Williamsburg, VA, USA, 2 Davis Heart & Lung Research Institute, Columbus, OH, USA. The recently introduced ‘‘probability density approach’’ to modeling local con- trol of CICR in cardiac myocytes [Williams et al. Biophys. J. 92(7):2311-28, 2007] and associated moment closure technique [95(4):1689-703, 2008] can re- produce whole cell voltage-clamp protocols high-gain Ca release that is graded with changes in membrane potential. This modeling formalism represents het- erogeneous local Ca signals in a population of diadic subspaces and junctional sarcoplasmic reticulum (jSR) depletion domains using a system of differential- algebraic equations for the time-evolution of the zeroth, first, and second mo- ments of probability density functions for jSR [Ca] jointly distributed with cal- cium release unit (CaRU) state. Here we show that a cardiac myocyte model that uses moment equations to represent heterogenous jSR Ca can exhibit Ca alternans when periodically stimulated by depolarizing voltage pulses, and makes predictions regarding the distribution of jSR [Ca] across a large popula- tion CaRUs as a function of stimulation frequency and cellular parameters such as the rate of diffusive transfer between network and junctional SR. Factors promoting alternating Ca responses in the moment closure model are analyzed and compared to analogous mechanism in a minimal ‘‘common pool’’ model with comparatively simple SR and PM fluxes. We derive load-release and re- lease-reuptake curves for both models, and investigate how model parameters influence these relations and the existence and stability of steady-state periodic Ca responses during repetitive depolarizing voltage pulses. Specifically, we find that increasing SR Ca leak, RyR sensitivity, and maximum release flux de- creases the steepness of load-release curves, shifts load-release curves to smaller SR loads, and increases the critical simulation frequency resulting in Ca alternans. 1415-Pos Board B259 New Insight Into Cardiomyocyte Ca Signaling Obtained By Fast Confocal Imaging Vyacheslav M. Shkryl, Lothar A. Blatter. Rush University Chicago, Chicago, IL, USA. With ultra-fast 1D-(x-t) and 2D-(x-y-t) confocal microscopy (Zeiss LSM 5 Live) we studied the spatio-temporal properties of Ca sparks and Ca transients. Ca sparks which originated from subsarcolemmal sarcoplasmic reticulum (SR) release sites in atrial myocytes were elongated in the longitudinal direction of the cell. Ca sparks corresponding to Ca release from non-junctional SR in atrial myocytes and junctional SR in ventricular myocytes were variable spa- tially with some events being symmetrical and others asymmetrical. Aniso- tropic sparks occurred in transverse as well as longitudinal direction. Ca sparks originating from non-junctional SR and recorded in line-scan (x-t) mode at 40,000 lines/s revealed a step-like appearance in space (time-depen- dent step-like increase of width from the point of origin) and amplitude during the activation phase of the spark. These steps in space and amplitude may rep- resent the sequential opening of individual ryanodine receptor (RyR) channels in a release cluster and support the notion that sparks represent Ca release from a group of RyRs. Mathematical analysis of global Ca transients recorded from field-stimulated ventricular myocytes at high temporal resolution al- lowed separation of Ca entry from Ca release flux. After the electrical stimulus a latency period of 2.5 ms was required to activate sarcolemmal Ca channels. SR Ca release was initiated with an additional delay of 3.0 ms. 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