Calcium Fluxes, Sparks, & Waves II 3106-Pos Board B261 Compensated Cardiomyocyte Ca 2D Homeostasis in a Rat Model of Diastolic Heart Failure A ˚ smund T. Røe 1 , Jan Magnus Aronsen 1 , Michael Frisk 1 , Ole M. Sejersted 1 , Ivar Sjaastad 1,2 , William E. Louch 1 . 1 University of Oslo, Oslo, Norway, 2 Oslo University Hospital, Oslo, Norway. In approximately half of heart failure patients, systolic function is near nor- mal while diastolic function is impaired. This condition, called diastolic heart failure (DHF), or heart failure with preserved ejection fraction (HFPEF), has been reported to result from reduced passive distensibility of the ventricle, which impairs filling. However, the contribution of altered Ca 2þ handling to impaired cardiomyocyte relaxation in this condition remains largely unin- vestigated. We examined this issue in a rat model of DHF following aortic stenosis. 6 weeks after aortic banding, cardiac function was characterized by echocardiography, and rats exhibiting systolic heart failure (left atrial di- ameter >5mm, left ventricular dilation and reduced fractional shortening) were excluded. Echo Doppler measurements revealed decreased peak early diastolic tissue velocity in DHF rats compared to sham-operated controls (6455 vs 8555 mm/s, P<0.05). Both heart and lung weights were increased in DHF animals, indicating hypertrophy and congestion. In isolated cardio- myocytes, Ca 2þ transients recorded by whole-cell fluorescence (fluo 4- AM) during field stimulation were of larger magnitude in DHF than control cells, across a range of frequencies (0.5-6 Hz). Ca 2þ transients also decayed more rapidly in DHF, and at high frequencies exhibited elevated SR content and a smaller increase in diastolic [Ca 2þ ]. Such augmentation of Ca 2þ ho- meostasis was consistent with an increase in both sarcoplasmic reticulum Ca 2þ reuptake and Ca 2þ extrusion in DHF cells. Confocal imaging of cells stained with di-8-ANEPPs indicated maintained t-tubule organization, con- sistent with an unaltered time to peak of the Ca 2þ transients. Thus, contrary to impaired Ca 2þ homeostasis widely reported in systolic heart failure, our results suggest that DHF develops despite improved Ca 2þ handling. We pos- tulate that other alterations such as increased passive ventricular stiffness or increased myofilament Ca 2þ sensitivity must impair diastolic function in this condition. 3107-Pos Board B262 Aortic Stenosis Triggers T-Tubule Growth in Human and Murine Cardiomyocytes Michael Frisk 1 , Guro F. Jølle 1 , Ida G. Lunde 1 , Biljana Skrbic 1 , Ole M. Sejersted 1 , Theis Tønnessen 1,2 , Ivar Sjaastad 1,2 , William E. Louch 1 . 1 University of Oslo, Oslo, Norway, 2 Oslo University Hospital Ullevaal, Oslo, Norway. Recent evidence indicates that cardiomyocyte t-tubule density and organiza- tion is highly malleable. T-tubules are lost and/or disorganized during heart failure, which causes de-synchronized Ca 2þ release from the sarcoplasmic reticulum and impaired contraction of the cardiomyocyte. We also recently reported that t-tubules are grown following SERCA ablation, which facili- tates increased trans-sarcolemmal Ca 2þ flux. The aim of this study was to in- vestigate t-tubule structure following aortic stenosis (AS) in patients and mice. Ventricular and atrial biopsies were obtained from patients undergoing aortic valve replacement (AS) or coronary artery bypass graft (control). Echocardiography was employed to exclude the presence of other cardiac diseases, and to evaluate the degree of valvular stenosis and hypertrophy. Biopsies were fixed and cryosectioned and t-tubules were visualized by con- focal microscopy following wheat germ agglutinin staining. T-tubules occu- pied 7.450.4% of the cross-sectional area of ventricular myocytes from AS patients, but only 5.350.6% of control cells (n=138, P<0.001). Similarly, t-tubule density was increased in atrial myocardium from AS patients com- pared to controls (5.950.6% vs. 3.950.3%, n=187, P<0.05). In addition, a larger fraction of cells displayed t-tubules in the AS group than in the con- trol group (52.9% vs. 30.4%, n=325, P<0.001). In mice with experimental AS (aortic banding), we observed that ventricular t-tubule density was in- creased relative to sham-operated controls 10 weeks after surgery (29.051.0% vs 24.450.7% of cross-sectional area, n=36, P<0.05). Analysis by fast Fourier transforms indicated that t-tubules remained well organized in AS cells. Consistent with this observation, synchrony of Ca 2þ release was maintained in enzymatically isolated AS cells loaded with fluo-4 and field stimulated at 1Hz. In conclusion, our observations indicate that AS promotes t-tubule growth in ventricular and atrial tissue of humans and mice. This ad- aptation may support compensated cardiomyocyte Ca 2þ homeostasis re- ported in this condition. 3108-Pos Board B263 Global and Local Effects of Intracellular pH on Ca 2D Waves in Rat Ventricular Myocytes Kerrie L. Ford 1 , Emma L. Moorhouse 1 , Mario Bortolozzi 1,2 , Richard D. Vaughan-Jones 1 . 1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, 2 Department of Physics G. Galilei, University of Padua, Padua, Italy. Arrhythmogenic Ca 2þ signalling may be influenced by alterations in in- tracellular pH (pH i ), as found in myocardial ischaemia. Here we character- ise the effects of altering pH i on Ca 2þ waves in rat isolated ventricular myocytes. Ca 2þ waves were induced by raising [Ca 2þ ] o , and imaged confocally using fluo-3. Parallel measurements of pH i were made using cSNARF-1. pH i changes were induced by superfusion of acetate (20-80mM) or trimethyl- amine (20mM). Reducing pH i acutely decreased (to 1656% of control) then increased (to 360562% by 60s) wave frequency. Inhibiting the sarcolemmal Na þ /H þ ex- changer (NHE) with 30mM 5-(N,N-dimethyl)amiloride suppressed this second- ary increase. A linear relationship was found between pH i and wave velocity: intracellular acidosis increased velocity up to 14053%. Acidosis also slowed wave decay and increased diastolic Ca 2þ , while alkalosis decreased diastolic Ca 2þ . A stable longitudinal pH i gradient (pH i 6.6-7.3) was generated in single myo- cytes by microperfusing half of the cell with normal Tyrode and half with 80mM acetate, using a double-barrelled micropipette. With NHE inhibited, a decrease in wave frequency was seen in the acidic microdomain and an in- crease in the more alkaline microdomain. Conversely, wave velocity was in- creased in acidic and decreased in alkaline microdomains. Thus, wave properties map onto a spatial gradient of pH i . Wave velocity may be influenced by multiple pH-dependent components. Computational modelling of Ca 2þ waves predicts that, at steady-state, sarco/en- doplasmic reticulum Ca 2þ ATPase inhibition decreases wave velocity, due to a fall in sarcoplasmic reticulum Ca 2þ content (Swietach et al, 2010). However, decreasing the k on of Ca 2þ buffers predicted an increase in wave velocity, sug- gesting the effect of pH i changes on velocity is multifactorial. We conclude that pH i heterogeneity locally regulates Ca 2þ waves, suggesting that it may contribute to the heterogeneity of Ca 2þ signalling observed in myo- cardial ischaemia. 3109-Pos Board B264 Fluid Pressure Triggers Action Potential and a Subsequent Transverse Ca 2D Wave via Na D –CA 2D Exchange Activated by Insp 3 Receptor- Mediated Local Ca 2D Wave in Rat Atrial Myocytes Joon-Chul Kim, Sun-Hee Woo. Chungnam National University, Daejeon, Korea, Republic of. We have previously shown that fluid pressure (FP) of ~16 dyn/cm 2 elicits longitudinal (L-) and transverse (T-) Ca 2þ waves in atrial myocytes. Here we characterized the FP-triggered T-waves and explored the underlying mechanisms involved using a 2-D confocal Ca 2þ imaging. A flow of pres- surized fluid (~16 dyn/cm 2 ) was applied onto single rat atrial myocytes using a microperfusion method. The FP-induced T-wave was characterized by a rapid synchronous peripheral Ca 2þ release followed by a delayed slower central Ca 2þ release. Most of the FP-induced T-waves contained preceding Ca 2þ increases. Pre-treatment of tetrodotoxin or clamping the membrane voltage eliminated the FP-induced T-waves, but not the preceding Ca 2þ sig- nals. The T-wave-preceding Ca 2þ signals were found to be (1) background increase with intermittent sparks, (2) crossing local wave, or (3) immature L-wave. The magnitudes of preceding Ca 2þ signals shown as the local waves (‘‘(1) and (2)’’) were significantly larger than those displayed as back- ground increases, were not different in the peripheral and central sites, and correlated with the rate of T-wave initiation. The preceding background Ca 2þ increases were larger in the periphery and irrelevant to the rate of T-wave initiation. The T-waves, preceded by only local Ca 2þ waves, had significantly larger Ca 2þ transients with high central releases compared with Ca 2þ transients during normal beats. Pre-exposure to KB-R7943, 2- APB, or U73122 suppressed the FP-induced T-waves that were preceded by local waves, but not those associated with background Ca 2þ increases. These results suggest that FP triggers action potentials and subsequent T- waves via both Ca 2þ -dependent and Ca 2þ -independent mechanisms, and that this Ca 2þ -dependent induction of action potential may involve activa- tion of inward Na þ -Ca 2þ exchange current due to InsP 3 receptor-mediated local Ca 2þ waves. Wednesday, February 6, 2013 603a