levels of miR-1 and miR-133 were significantly increased in HF myocytes compared to controls (2 and 1.6 fold accordingly). Western blotting showed that PP2A regulatory (b56alpha) and catalytic subunits, specific targets of miR-1 and miR-133 validated by luciferase-reporter assay, were decreased in HF cells. Analysis using phospho-specific antibodies confirmed that RyR2 phosphorylation at Ser-2814 was significantly increased in HF myo- cytes compared to controls. CaMKII inhibitory peptide reduced the frequency of spontaneous Ca waves in paced current-clamped HF myocytes to low con- trol values. These finding suggest that altered levels of major muscle-specific microRNAs contribute to abnormal RyR2 function in HF by depressing local- ized phosphatase activity to the channel, thus leading to excessive phosphor- ylation of RyR2s. 3045-Pos Board B150 Voltage-Dependent Anion Channel 2 modulates Resting Calcium Sparks, but not Action Potential-Induced Global Calcium Signaling in Cardiac Myocytes Krishna P. Subedi, Joon-Chul Kim, Moonkyung Kang, Yeon-Soo Kim, Sun-Hee Woo. Voltage-dependent anion channels (VDACs) are pore forming proteins pre- dominantly found in the outer mitochondrial membrane and is thought to transport calcium ion (Ca 2þ ). In this study, we have investigated the possible role of type 2 VDAC (VDAC2) in cardiac Ca 2þ signaling and Ca 2þ sparks using a lentiviral knock-down (KD) technique and two-dimensional confocal Ca 2þ imaging in immortalized autorhythmic adult atrial cells, HL-1. We con- firmed high expression of VDAC2 protein in ventricular, atrial and HL-1 cells using Western blot analysis. Infection of HL-1 cells with VDAC2-targeting lentivirus reduced the level of VDAC2 protein to ~10%. Comparisons of au- torhythmic Ca 2þ transients between wild type (WT) and VDAC2 KD cells showed no significant change in the magnitude, decay, and beating rate of the Ca 2þ transients. Caffeine (10 mM)-induced Ca 2þ release, which indicates sarcoplasmic reticulum (SR) Ca 2þ content, was not altered by VDAC2 KD. Interestingly, however, the intensity, width, and duration of the individual Ca 2þ sparks were significantly increased by VDAC2 KD in resting condi- tions, with no change in the frequency of sparks. These results suggest that VDAC2 may suppress focal Ca 2þ releases through ryanodine receptors in atrial myocytes under resting conditions. The results also indicate that VDAC2 may not regulate action potential-induced global Ca 2þ signaling and SR Ca 2þ loading. 3046-Pos Board B151 African Trypanosomes Increase Calcium Wave Frequency in Isolated Adult Rat Cardiomyocytes via Secretion of Cathepsin L Elspeth B. Elliott, Liam J. Morrison, Hisashi Hasumi, Christopher M. Loughrey. African trypanosomes are blood-borne extracellular parasites which have recently been linked to cardiac dysfunction in ~70% of sleeping sickness patients. Although this may result from an indirect effect of the parasite (e.g. myocarditis), a direct effect of the parasite on the heart has not been investigated. Adult rat cardiomyocytes were incubated with trypanosome growth media containing Trypanosoma brucei Lister427 (30min). A popula- tion assay assessed the percentage of cells demonstrating Ca 2þ waves within a 1min period. Incubation with live trypanosomes led to a significant increase in the percentage of cells demonstrating Ca 2þ waves (54.852.8% vs. 79.255.1%; media vs. live trypanosomes, P<0.05; n=4294 and 3006 cells re- spectively). This effect was maintained when cells were incubated with super- natant (trypanosomes removed from media by centrifugation) (77.352.9%; n=2131 cells). Separate experiments showed the supernatant effect was lost upon boiling (83.751.8% vs. 66.352.4%; supernatant vs. boiled supernatant, P<0.05; n=527 and 612 cells respectively). Results were confirmed in Fur- a2AM loaded, field stimulated (1Hz) rat cardiomyocytes perfused with media (37 o C). Following 4 min supernatant perfusion, the frequency of Ca 2þ waves in the inter-stimuli interval was significantly increased (0.0250.01 vs. 0.4450.07 waves/s; media vs. supernatant, P<0.05; n=10). Since the parasite induces a similar phenomenon in brain mono-epithelial cells via cathepsin-L cysteine protease, we examined the role of cathepsin-L in the above effect on cardiomyocytes. In separate experiments, supernatantþK11777 (specific inhibitor of cathepsin-L) completely abolished the ability of supernatant to increase Ca 2þ wave probability (56.355.1 vs. 49.155.7%; media vs. super- natantþK11777, P>0.05), whereas CA074 (specific inhibitor of cathepsin-B) had no effect on Ca 2þ wave frequency. These data suggest trypanosomes in- teract with cardiomyocytes leading to increased Ca 2þ wave production via cathepsin-L. This may contribute to the cardiac abnormalities observed in patients with trypanosomiasis. 3047-Pos Board B152 Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes E. Michelle Capes, Randall E. Loaiza, Roberto Ramos, Jianhua Zhang, Timothy J. Kamp, Hector H. Valdivia. Fibroblasts from human skin biopsies can be reprogrammed into pluripotent stem cells (iPSC), which can then be coaxed to differentiate into myocytes with cardiac-specific properties (iPSC-CMs). The field of iPSCs is still in its infancy, but it is increasingly clear that the excitation-contraction coupling (ECC) machinery of differentiating CMs undergoes proportionally incremental complexity and it remains to be seen whether it reaches complete maturity in cultured cells. We used patch-clamp and confocal Ca 2þ imaging for a compar- ative assessment of ECC in human iPSC-CM and adult cardiomyocytes. In the latter, entry of Ca 2þ through the L-type Ca 2þ channel (I Ca ) triggers rapid, uni- form release of Ca 2þ from the sarcoplasmic reticulum (SR) via CICR. In iPSC- CMs at early stages of differentiation, the current-voltage relationship for I Ca is remarkably similar to that of adult cardiomyocytes, indicating that the appear- ance of a ‘‘trigger’’ for contraction is an early event in the ontogenesis of ECC that doesn’t hinder efficient generation of Ca 2þ signals. However, primitive iPSC-CMs commonly exhibit a poorly developed SR, as assessed by their var- iegated response to caffeine and their great dependence on extracellular Ca 2þ for contraction. Cells are mostly rounded and t-tubules are absent. As a result, [Ca 2þ ] i transient waveforms appear non-uniform and start at the periphery of the cell, as is expected of a Ca 2þ front with focal initiation that propagates later to the interior of the cell. At more advanced stages of differentiation, iPSC- CMs display fairly uniform Ca 2þ fronts, suggesting fast propagation of external Ca 2þ signals to the interior of the cell. Thus, by this coarse functional estimate, it is expected that iPSC-CMs become accurate models of cardiomyopathies at late stages of differentiation, but the developmental characteristics of ECC is unclear and warrants a systematic approach, which we are currently performing. 3048-Pos Board B153 Impaired Calcium Signaling Refractoriness Contibutes to Increased Rate of Diastolic Calcium Waves in Myocytes from Post-Myocardial Infarction Hearts Andriy E. Belevych, Dmitry Terentyev, Radmila Terentyeva, Hsiang-Ting Ho, Cynhtia A. Carnes, George E. Billman, Sandor Gyorke. Spontaneous Ca 2þ waves (SCWs) are recognized as important contributors to triggered arrhythmia. SCWs waves are thought to arise when [Ca 2þ ] SR reaches a certain threshold level, which might be reduced in cardiac disease as a conse- quence of sensitization of ryanodine receptors (RyR2s) to luminal Ca 2þ . We investigated the mechanisms of SCW generation by simultaneous measure- ments of cytosolic and luminal Ca 2þ in myocytes from normal and diseased hearts using a canine model of post-myocardial infarction (MI) tachyarrhyth- mia. The frequency of SCW, recorded during periodic pacing in the presence of b-adrenergic receptor agonist isoproterenol, was significantly higher in MI myocytes than in control. Rather than occurring at once upon reaching a final [Ca 2þ ] SR , SCWs arose with a distinct time delay from the attainment of the maximum [Ca 2þ ] SR in both experimental groups. While the rate of [Ca 2þ ] SR recovery following the SR Ca 2þ release was similar between the two myocyte types, the maximally attainable [Ca 2þ ] SR was lower, and the latency to SCW was shorter in MI myocytes compared to control. Both phosphorylation at the CAMKII site Ser-2814 and the level of oxidized thiols were higher in RyR2s from MI hearts than in control. The CAMKII inhibitor, KN93, or the reducing agent, mercaptopropionylglycine, reduced SCW frequency in MI my- ocytes. The MI-related alterations in myocyte Ca 2þ cycling were mimicked by the RyR2 agonist, caffeine. These results indicate that attainment of a certain threshold [Ca 2þ ] SR is not a sufficient condition for the generation of SCWs and that Ca 2þ signaling refractoriness that develops following release critically influences SCW occurrence in the diastolic period. We conclude that shortened Ca 2þ signaling refractoriness due to RyR2s phosphorylation and oxidation is responsible for the increased rate of SCWs observed in MI myocytes. 3049-Pos Board B154 Inositol 1,4,5 Triphosphate (IP3) Receptors Activate Type 1 ryanodine Receptors to Mediate Ca 2D Sparks Signaling in Adult Mammalian Skele- tal Muscle Andoria Tjondrokoesoemo, Na Li, Noah Weisleder, Jianjie Ma. Ca 2þ sparks are the elemental event of Ca 2þ induced Ca 2þ release (CICR) that originate from clustered ryanodine receptor Ca 2þ release channels (RyR1) in mammalian striated muscles. Previously we found that application of transient osmotic stress to the intact skeletal muscle leads to a robust Ca 2þ spark re- sponse that is restricted to the periphery of sarcolemmal membrane. Here we Wednesday, March 9, 2011 561a