Dantrolene Inhibition of Skeletal Muscle RYR in the ... - Cell Press

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Feb 28, 2016 - for malignant hyperthermia (MH). Our group recently reported that dantrolene inhibition of RyR1 and RyR2 required calmodulin (CaM)1.
Sunday, February 28, 2016

Excitation-Contraction Coupling I 499-Pos Board B279 Ryanodine Receptor Sensitivity Governs the Stability and Synchrony of Local Calcium Release during Cardiac Excitation-Contraction Coupling Andrew P. Wescott1, M. Saleet Jafri1,2, W. Jonathan Lederer1, George S.B. Williams1. 1 BioMET, University of Maryland Baltimore, Baltimore, MD, USA, 2School of Systems Biology, George Mason University, Manassas, VA, USA. Synchronous coordination of calcium-induced calcium release (CICR) across thousands of calcium release units (CRUs) within the cardiomyocyte is critical for normal excitation-contraction coupling (ECC). Here we utilize a novel ‘‘local control’’ mathematical model of ECC, designed to simulate a mouse cardiomyocyte, to investigate physiological and pathophysiological calcium (Ca2þ) signaling. The model contains 20,000 independent CRUs each composed of 6 stochastically gated L-type Ca2þ channels (LCCs) in the transverse tubule membrane that are positioned across a local ‘‘dyadic subspace’’ from a junctional sarcoplasmic reticulum (JSR) compartment containing a cluster of 50 stochastically gated ryanodine receptors (RyR2s). Action potential depolarization of the sarcolemmal membrane activates LCC openings which elevate subspace [Ca2þ] ([Ca2þ]ds) across each of the cell’s 20,000 CRUs. This rise in [Ca2þ]ds promotes RyR2 opening to initiate the fundamental element of triggered Ca2þ release, the Ca2þ spark. The mechanistic design and true SR Ca2þ pump/leak balance displayed by our model allows us to quantify ECC fidelity and Ca2þ spark fidelity (the probability that a LCC opening or RyR2 opening induces a Ca2þ spark, respectively). We study the effect of excess SR Ca2þ leak in the context of ‘‘catecholaminergic polymorphic ventricular tachycardia (CPVT)’’ caused by the R33Q mutation in calsequestrin (CASQ2). CPVT is simulated by increasing RyR2 sensitivity to [Ca2þ]i and reducing JSR Ca2þ buffering capacity to levels identified by prior studies of the CASQ2-R33Q ‘‘knock-in’’ mice. Our model effectively reproduces both normal and arrhythmogenic CPVT RyR2 [Ca2þ]i sensitivity, local Ca2þ spark activity, and global Ca2þ signals. Under CPVT conditions enhanced RyR2 open probability is critical for the development of ‘‘unstable’’, quiescent Ca2þ sparks that fail to terminate robustly as well as asynchronous systolic Ca2þ spark activity and increased diastolic SR Ca2þ leak. 500-Pos Board B280 Dantrolene Inhibition of Skeletal Muscle RYR in the Presence of CAM YE W. Oo, M.S. Imtiaz, D.F. vanHelden, D.R. Laver. Discipline of Human Physiology, The University of Newcastle, Newcastle, Australia. Dantrolene is a muscle relaxant that has been used clinically as the treatment for malignant hyperthermia (MH). Our group recently reported that dantrolene inhibition of RyR1 and RyR2 required calmodulin (CaM)1. However, nothing more is known about the properties of dantrolene inhibition of RyR1 on single channels. Here, we characterized dantrolene inhibition on RyR1 in the presence of exogenous CaM at various cytosolic [Ca2þ] and in the presence of other activators: ATP, halothane and domain peptide, DP4. RyR1 was isolated from rabbit skeletal muscle and incorporated into artificial lipid bilayers and their open probability, Po, was measured using single channel recording. Dantrolene caused inhibition of RyR1 and RyR2, but when CaM was presence in the cytoplasmic bath. In the presence of cytoplasmic [Ca2þ] (100 nmol/l) and 2 mmol/l ATP, dantrolene inhibited both RyR1 and RyR2 with identical hyperbolic doseresponses with IC50 of 0.16 5 0.03 mmol/l and with saturating Po of 52 5 4 % of control. RyR1 has a bell-shaped cytoplasmic Ca2þ activation curve with half activation at 1.75 5 0.62 mmol/l and half inhibition at 0.21 5 0.02 mmol/l and a peak Po = 0.94 5 0.06. Dantrolene reduced Po in sub-activating and inhibiting [Ca2þ] but failed to reduce peak Po. Dantrolene caused 50-60% reduction in Po of RyR1 activated by cytoplasmic Ca2þ alone (100 nmol/l) or in conjunction with halothane (5 mmol/l) or DP4 (10 mmol/l). To conclude, dantrolene inhibits RyR1 activity by 1) decreasing RyR1 sensitivity to Ca2þ activation, 2) increasing sensitivity to Ca2þ inhibition and 3) decreasing channel activation halothane or by 4) reducing the effect of inter-domain disruption in the RyR1 protein. 501-Pos Board B281 Novel Compounds Inhibit Calmodulin Deficient RyR2 Activity and Arrhythmias in a CPVT Mouse Model Robert C. Klipp1, Na Li2, Qiongling Wang2, Martha Sibrian-Vazquez3, Robert M. Strongin3, Xander H.T. Wehrens2, Jonathan J. Abramson1. 1 Physics, Portland State university, Portland, OR, USA, 2Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX, USA, 3 Chemistry, Portland State university, Portland, OR, USA. The cardiac ryanodine receptor (RyR2) plays a key role in excitationcontraction (EC) coupling. Mutations in RyR2 are known to be linked to the

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arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), a deadly disease which is characterized by a leak of calcium from sarcoplasmic reticulum and a decrease in calmodulin (CaM) binding. A novel drug, 84-F2, is shown to inhibit arrhythmias in RyR2-R176Q heterozygous CPVT mouse hearts (2.5 mg/kg), decrease spark frequency in cells derived from CPVT mice (IC50 = 35nM), and inhibit RyR2 single channel activity at low nanomolar concentrations (IC50 = 11nM). When CaM is added back, 84-F2’s ability to inhibit channel activity is suppressed ~100 fold. We propose that this new drug decreases arrhythmias by binding to the CaM deficient RyR2, but does not affect normal EC coupling because of the presence of CaM. This work shows for the first time a class of drugs whose inhibitory effects are dependent upon the removal of CaM from RyR2. Supported by NIH 2R42HL114206 and R41-HL129570 to Elex Biotech, LLC. 502-Pos Board B282 S4-S5 Linker Regulates RyR2 Channel Gating through Multiple Interactions Takashi Murayama1, Nagomi Kurebayashi1, Haruo Ogawa2, Junji Suzuki3, Kazunori Kanemaru3, Masamitsu Iino3, Takashi Sakurai1. 1 Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan, 2Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, 3Department of Cellular and Molecular Pharmacology, The University of Tokyo, Tokyo, Japan. Type 2 ryanodine receptor (RyR2) is a Ca2þ release channel in the sarcoplasmic reticulum of cardiac muscle and plays an important role in excitation-contraction coupling. Although recent cryo-EM studies provided near-atomic structures of type 1 ryanodine receptor (RyR1), molecular mechanism of the channel opening remains largely unknown. The S4-S5 linker (S45L) is an a-helical structure connecting the S4 and S5 transmembrane segments and mediates signal transmission in a wide variety of channels. To address the role of S45L in the RyR2 channel gating, we systematically mutated residues in S45L (Thr4751-Asn4762) and neighboring transmembrane segments (S5 and S6). The mutant RyR2 was stably expressed in HEK293 cells, and the channel activity was investigated by ER luminal Ca2þ measurements and [3H]ryanodine binding. Using high sequence identity between RyR1 and RyR2 (65% in the whole molecule and 93% in the core channel domain after S4), we constructed structural model of RyR2 based on the structures of RyR1, and the phenotypes of mutants were interpreted by the model. Plausible interactions between amino acid residues in S45L and neighboring domains that may regulate channel gating will be discussed. 503-Pos Board B283 Spontaneous and Voltage-Activated Elementary Calcium Release Events in Intact Skeletal Muscle Fibers Expressing the Embryonic CaV1.1 Splice Variant Beatrix Dienes1, Ja´nos Vincze1, Pe´ter Szentesi1, Nasreen Sultana2, Berhnard E. Flucher2, La´szlo´ Csernoch1. 1 Department of Physiology, University of Debrecen, Debrecen, Hungary, 2 Medical University Innsbruck, Innsbruck, Austria. The adult isoform of voltage-gated L-type calcium channel (CaV1.1a) opens slowly at strong depolarizations, its contribution to the calcium influx during an action potential is negligible in skeletal muscle. In contrast, calcium influx through the embryonic splice variant (CaV1.1e) substantially contributes to depolarization-induced calcium transients as this isoform displays an altered voltage-dependence and gating kinetics as compared to CaV1.1a. Utilizing a genetically modified mouse model (CaV1.1aE29), which exclusively expresses the embryonic CaV1.1e variant in adult muscle, spontaneous or depolarizationevoked calcium release events were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum brevis, using the fluorescent calcium probe Fluo-8 and the Zeiss Live fast confocal microscope equipped with an Axon Multiclamp 700b. The events occurred at preferred locations as the distribution of spark number in the given position was different from the theoretical Poisson distribution which represents random event occurrence. Calcium events with both short (spark-like, duration = 46.051.5 ms, n = 125) and long (ember-like, duratio n= 473514 ms, n = 218) duration were present on the same cell. In addition, events with complex kinetics, including late openings within an event also appeared. Voltage-activated (500 ms long pulses to the threshold of SR calcium release) with similar properties were also observed. 504-Pos Board B284 Characterization of the STAC3-CaV1.1 Interaction in Skeletal Muscle Excitation-Contraction Coupling Marta Campiglio, Bernhard E. Flucher. Physiology and medical Physics, Medical Innsbruck University, Innsbruck, Austria. Skeletal muscle EC coupling critically depends on the physical interaction between the voltage-gated calcium channel CaV1.1 (DHPR), acting as voltage