1568-Pos Board B338 In Vitro Characterization of a Novel N-Terminal CPVT RyR Mutation Alexandra Zahradnikova, Jr. 1 , Patricia Neco 1 , Eric Morel 1 , Alejandro Dominguez Rodriguez 1 , Spyros Zissimopoulos 2 , F. Anthony Lai 2 , Jean-Pierre Benitah 1 , Esther Zorio 3 , Ana Maria Gomez 1 . 1 INSERM U-769, Cha ˆtenay-Malabry, France, 2 Cardiff University School of Medicine, Cardiff, United Kingdom, 3 Hospital Universitario La Fe, Valencia, Spain. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an ar- rhythmogenic disease characterized by stress-triggered syncope and sudden death. Most of the CPVT mutations are concentrated in RyR ‘‘hotspots’’: the C terminus, the central and the N terminal domains. Although the phenotypic manifestations are common, it is likely that the intrinsic mechanism of RyR dysfunction varies with the location of the mutation within the RyR. Previ- ously, we characterized RyR dysfunction in a CPVT mutation located at the C terminal domain, identifying an increased cytosolic Ca 2þ sensitivity as the causative of a gain-of-funcion defect. Recently, we have identified a mutation in a family with CPVT syndrome, which is located in the N terminal portion: RyR2 R420Q . To analyze the function of the mutated RyR channel, we have cre- ated an expression plasmids of RyR2 R420Q and RyR2 WT . Heterologous expres- sion in HEK cells showed decreased caffeine sensitivity, suggesting loss-of- function defect. In order to be able to further characterize this mutation in a car- diac context, we expressed both RyR2 R420Q and RyR2 WT in cultured neonatal rat cardiac myocytes and analyzed the amplitude and frequency of spontaneous [Ca 2þ ] i transients and Ca 2þ sparks by confocal microscopy. We found that the spontaneous [Ca 2þ ] i transients were more frequent but of lower amplitude in RyR2 R420Q expressing cells. Further experiments are being conducted to fully understand the underlying mechanism. 1569-Pos Board B339 Regulation of Mitochondrial Ca 2D Uptake by Mitochondrial Ryanodine Receptor in Control and Malignant Hyperthermia Mouse Heart Polina Gross 1 , Niina Sokolova 2 , Shi Pan 1 , Gisela Beutner 3 , Shey-Shing Sheu 1 . 1 Thomas Jefferson University, Philadelphia, PA, USA, 2 Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia, 3 University of Rochester, Rochester, NY, USA. Malignant hyperthermia (MH) is a severe hypermetabolic response triggered by exposure to volatile anesthetics and caused by mutations in the genome of ryanodine receptor subtype 1 (RYR1) in the skeletal muscle. The presence of mitochondrial ryanodine receptor (mRYR) in heart, which behaves immuno- logically and pharmacologically like RyR1, raises the question of its primary cardiac pathological role in MH. To address this, we used genetically modified micecontaining RyR1 with a single point mutation at the position 522 (tyrosine versus serine; YS mice, obtained from Dr. Susan Hamilton, Baylor College of Medicine) and wild type (WT) mice. Ca 2þ uptake of isolated heart mitochondria from both animal groups was measured (N=3) using ArsenazoIII. Our data show, that in 1 year old YS mice, the total free Ca 2þ load tolerated before full mitochondrial permeability transition pore (mPTP) opening was lower (275.22542.4 nmol free Ca 2þ / mg protein) compared to WT (474.77565.7 nmol free Ca 2þ / mg protein). The time constant (T), calculated from the velocity of Ca 2þ uptake during the first 2 minutes after applying a 5mM Ca 2þ pulse was not significantly different in YS than in WT. However, significant differences were observed using RYR1 specific inhibitors (10mM dantrolene and 10mM azumolene). In presence of these inhibitors, WT showed a prolonged T (383.09541.65sec with dantrolene and 305.75538.52sec with azumolene) suggesting that mRYR was inhibited. Sur- prisingly, YS showed less inhibition due to the difficulty of blocking leaky chan- nel (265.55526.07sec and 202.56531.45sec respectively). Measuring the membrane potential with TMRE showed that baseline fluores- cence of YS mitochondria was significantly lower, indicating that these mito- chondria are depolarized (1.6150.02a.u. versus 1.9350.02a.u. in WT, P<0.05). Depolarized mitochondrial membranes due to leaky mRYR suggest a primary role of this protein in heart pathology in MH. 1570-Pos Board B340 Dantrolene-Sensitive Mitochondrial Ca2D Uptake under Mitochondrial Ca2D Uniporter Inhibition Shi Pan, Nadan Wang, Polina Gross, Jin O-uchi, Shey-Shing Sheu. Thomas Jefferson University, Philadelphia, PA, USA. Intracellular Ca2þ is vital for cell physiology. Disruption of Ca2þ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. The central role of mitochondrial Ca2þ is to regulate energy produc- tion and shape the amplitude and duration of Ca2þ transients. In responding to physiological Ca2þ pulses, mitochondria take up Ca2þ through the activation of influx mechanisms in the inner membrane, such as mitochondrial Ca2þ uni- poter (MCU), and mitochondrial ryanodine receptor (mRyR). Dantrolene is a pharmacological compound used clinically for the treatment of malignant hyperthermia by blocking RyR. Our recent studies have shown that mRyR is sensitivity to dantrolene, but not to ruthenium red 360 (Ru360), which is a known inhibitor of MCU. Therefore, we studied the mechanisms for mito- chondrial Ca2þ uptake in the existence of MCU inhibitor Ru360 and mRyR inhibitor dantrolene. Cardiac mitochondria isolated from C57BL/6 mice were incubated with dantro- lene (10 mM), Ru360 (50 nM), or both Ru360 and dantrolene. Mitochondrial Ca2þ uptake was measured using a fluorometer with Calcium Green added in the assay solutions. Ru360 significantly blocked mitochondrial Ca2þ up- take. However, there still existed a small but statistically significant transient Ca2þ uptake component. This transient Ca2þ uptake component was inhibited by dantrolene. Our results demonstrate a dantrolene-sensitive Ca2þ uptake mechanism in car- diac mitochondria. This Ca2þ uptake mechanism appears to be faster and tran- sient in comparison to that of MCU. 1571-Pos Board B341 Physical Interaction of RyR2 and VDAC2 as a Molecular Architecture for the Coupling Between Sarcoplasmic Reticulum and Mitochondria in Cardiomyocytes Choon Kee Min 1 , Dong Rim Yeom 1 , Kyung-Eun Lee 2 , Hye Sung Jeon 2 , Do Han Kim 1 . 1 GIST, Gwangju, Korea, Republic of, 2 KIST, Seoul, Korea, Republic of. Inter-organellar physical coupling between mitochondria and sarcoplasmic re- ticulum (SR) and the presence of high Ca 2þ microdomain for Ca 2þ transfer into mitochondria have been reported in cardiomyocytes (Garcia-perez et al., J. Biol. Chem., 2008). However, the molecular components involved in the phys- ical coupling remain to be elucidated. In the present study, we identified VDAC2 as a putative binding partner for RyR2 in SR by the bacterial two- hybrid screening. The interaction of VDAC2 with RyR2 was further confirmed by GST-pull down and co-immunoprecipitation assays. As tethering structures between SR and mitochondria was detected in cardiomyocytes (Boncompagni et al., Mol. Biol. Cell., 2009), we identified a close apposition between mito- chondria and SR in HL-1 cells by electron microscopic study. With the immuno-gold labeling strategy, furthermore, the co-localization of VDAC2 with RyR2 at the junction between SR and mitochondria was revealed at sub-micrometer resolution. In adult ventricular cardiomyocytes, the association of VDAC2 and RyR2 was detected at the sub-sarcolemmal region according to immunocytochemical study and differential centrifugations. To investigate the roles of VDAC2 in cardiomyocytes, we attempted to use adenovirus-based shRNA system to knock down VDAC2 gene expression. Under electrical field stimulation (1Hz), the diastolic [Ca 2þ ] increased significantly in VDAC2 knockdown cells without alteration of Ca 2þ transient amplitude and Ca 2þ up- take rate. Taken together, the present study suggests that VDAC2 and RyR2 are the molecular components for the linkage between mitochondria and SR, and regulate Ca 2þ signaling in cardiomyocytes. (Supported by Korea MEST NRF grant (20110002144), the 2011 GIST Systems Biology Infrastructure Establish- ment Grant and KISTI-KREONET) Calcium Fluxes, Sparks, and Waves II 1572-Pos Board B342 Effect of Ryanodine Receptor Refractoriness on the Induction of Calcium Alternans Enric Alvarez-Lacalle 1 , Inmaculada R. Cantalapiedra 1 , Angelina Pen ˜aranda 1 , Leif Hove-Madsen 2 , Blas Echebarria 1 . 1 Universitat Polite `cnica de Catalunya, Barcelona, Spain, 2 Cardiovascular Research Centre CSIC-ICCC, Hospital de Sant Pau, Barcelona, Spain. A numerical model of the rabbit ventricular cardiomyocyte is developed, that properly reproduces calcium alternans at high pacing frequency and experi- mental postrest-potentiation. We show that calcium alternans may appear due to the alternation in the number of RyR2 ready to open before each beat and not, despite the fact they are present, to oscillations in the SR Ca load. This mechanism is present when activation rates of RyR2 are low and is also favored by a slowing of the RyR2 recovery time from inactivation. Generally, changes in RyR2 refractoriness lead to alternans via two different mechanisms, (the already established) SR Ca2þ load and (a new one) recovery form inacti- vation. Drugs or mutations which target RyR2 refractoriness should be arryth- mogenic via the activation of one of those mechanisms if they decrease the activation or inactivation rate. 308a Monday, February 27, 2012