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Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease
Wednesday, February 15, 2017 2657-Pos Board B264 Tonic Activation of Extrasynaptic NMDA Receptors Decreases Neuronal Excitability in Alzheimer’s Disease David Gall1, Antonio Lobo-Antunes2, Genevie`ve Dupont2. 1 Laboratoire de Physiologie et Pharmacologie (CP604), Universite´ Libre de Bruxelles, Bruxelles, Belgium, 2Unite´ de Chronobiologie The´orique (CP231), Universite´ Libre de Bruxelles, Bruxelles, Belgium. Amyloids b (Ab) are a hallmark of Alzheimer’s disease. They affect the communication between neurons. They can also bind to neuronal targets and thereby affect both intracellular signalling and neuronal electrical activity. During the onset of Alzheimer’s disease, a positive feedback loop between Ab40/ 42 and cytosolic calcium is thoughtto accelerate the progression of the disease. If intracellular calcium and Ab reinforce themselves through this mechanism, one would expect that the neurons targets of Ab may display an altered electrical activity caused by the increase in cytoplasmic calcium as it is known that there is a tight coupling between calcium dynamics and the electrical excitabilty. The aim of this work is to test this assumption when considering one of the privileged target of Ab, the activity of extrasynaptic NMDA receptors. Our theoretical model is a simple description of neuronal electrical activity based on the Hodgkin-Huxley like formalism, including a term that corresponds to the activity of the NMDA receptor and a cytosolic calcium compartment. When the tonic activity of extrasynaptic NMDA receptors is increased, neurons are less excitable. This is a counterintuive result as NMDA receptors exert an excitatory effect. Further analysis show that this inhibitory effect is due to the activation of calcium-dependent potassium channels, which hyperpolarize the neurons. Activation of extrasynaptic NMDA receptors also provokes a marked increase in intracellular calcium concentration, thus reinforcing the feed-forward relation between Ab production and calcium. 2658-Pos Board B265 Regulation of Axon Growth by Alpha 7 Nicotinic Receptor Calcium Transients at the Growth Cone E. Bak, J. Jedrzejewska-Szmek, J. King, K. Blackwell, N. Kabbani. Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA, USA. Cell surface receptors of the growth cone (GC) transmit extracellular information that is essential for synaptogenesis and proper brain wiring. Various external cues including neurotransmitter gradients serve to modulate the turning, extension, and retraction of the GC by targeting intracellular calcium signaling pathways. Both the magnitude of the intracellular calcium rise as well as its source encode specific information leading to cytoskeletal remodeling during axon growth or retraction. We have shown that calcium conducting alpha7 nicotinic acetylcholine receptor (a7nAChR) channels bind Gaq thereby promoting IP3 receptor mediated calcium store release during nAChR channel desensitization. Here we examine the role of a7 nAChR-mediated calcium transients in neurite growth and retraction using a stochastic reaction-diffusion model of calcium gradients, cAMP, and Gaq pathways within the GC. The model allows for prediction of intracellular calcium dynamics via the entry of calcium through the a7 nAChR, activation of voltage gated channels channels, and the metabotropic signaling properties of the a7nAChR on local ER. We show that the identity of the calcium source impacts the dynamics of non-linear interactions between a cohort of calcium sensitive effectors such as PP2B and PP1, CaMKII, PKA, and calpain. Our model begins to explain experimental observations on neurite growth in cultured PC12 cells and hippocampal neurons suggesting non-monotonic dependence of structural growth on calcium levels, where both high and low calcium can inhibit growth. Elucidating the mechanisms of calcium signaling within the GC yields a better understanding of synaptic growth and plasticity, as well as an opportunity for fostering regeneration. 2659-Pos Board B266 Inorganic Polyphosphate Protects Neurons against Glutamate-Induced Excitotoxicity Marta Maiolino1, Vincenzo Lariccia2, Salvatore Amoroso2, Plamena R. Angelova1, Andrey Y. Abramov1. 1 Molecular Neuroscience, UCL Institute of Neurology, London, United Kingdom, 2Biomedical Science, UNIVP Universita’ Politecnica delle Marche, Ancona, Italy. Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative diseases. Loss of calcium homeostasis and mitochondrial dysfunction are the key mediators of glutamate induced cell death. Recently, we found that inorganic polyphosphate (Poly P) can act as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in regulation of neuronal activity remains still undefined. Considering the number of studies which demonstrate the close interaction between neurons and glia in physiology and pathology, we studied the effect of the polyP on glutamate induced calcium signal in neurons, in
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physiological and pathological conditions. We used fluorescence imaging to measure mitochondrial membrane potential and intracellular calcium concentration ([Ca2þ]i) in primary cultures of hippocampal or cortical neurons (10-15 days in vivo), using Rhodamine123 and Fura-ff respectively. Application of glutamate (50-100mM) causes a stereotypical response consisting of an initial transient spike in [Ca2þ]i followed by a secondary increase in [Ca2þ]i which is coincident with a delayed mitochondrial collapse. Pretreatment of the neurons and astrocytes with different length of polyP significantly reduced the number of cells with a secondary delayed calcium deregulation induced by high concentration of glutamate. Moreover, glutamateinduced mitochondrial depolarization was also prevented by PolyP addition. Importantly, long chain PolyP successfully protected cells against glutamate induced cell death. Thus, polyP protects neurons against glutamate-induced excitotoxicity by reduction of the calcium overload and restoring mitochondrial function.
Intercellular Calcium Channels and Calcium Sparks and Waves II 2660-Pos Board B267 ATP Release through Gap Junction Hemichannels Increases Ca2D Spark Occurrence via P2Y Purinoceptor Signaling in Rat Ventricular Myocytes under Shear Stress Jun Wang, Joon-Chul Kim, Sun-Hee Woo. College of Pharmacy, Chungnam National University, Daejeon, Korea, Republic of. Shear stress in ventricles increases under pressure/volume overload caused by valve diseases, heart failure, and hypertension and enhances Ca2þ transients in ventricular myocytes. We have previously proposed that the shear-mediated Ca2þ transient increase is in part due to sensitization of Ca2þ release sites through ROS generation by NADPH oxidase (NOX) and NO synthase (NOS). Here, we investigated remaining mechanism for the activation of Ca2þ release sites under shear stress in rat ventricular myocytes. Shear stress of ~16 dyn/cm2 was applied onto single cells using micro-jet apparatus. Two-dimensional confocal Ca2þ imaging was performed at 30 Hz. The frequency of resting Ca2þ sparks was immediately increased to ~180% and further increased to ~250% by prolonged exposure (20 s). Pretreatment of cells with inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor 2-APB (2 mM) or protein kinase C (PKC) inhibitor chelerythrine (2 mM) partly (about 50%) suppressed both immediate and prolonged shear effects on the spark occurrence. Blockade of P2 purinoceptors (30 mM suramin) almost completely suppressed shear-induced spark enhancements. Inhibition of phospholipase C (PLC) using U73122 (5 mM) significantly suppressed (~80%) shear-induced Ca2þ spark increases. Pretreatment of P2Y1 receptor antagonist MRS2179 (400 nM) diminished immediate and late increases in spark occurrence during shear stimulation by 60-70%. Inhibition of gap junction hemichannels using carbenoxolone (50 mM) or external treatment of ATP metabolizing apyrase (2 units/ml) eliminated the stimulatory effects of shear stress on the spark occurrence. Consistently, luciferin-luciferase assay revealed ATP release from these myocytes by shear stress within 2 s. These results suggest that shear stress may enhance Ca2þ spark occurrence partly via activation of P2Y-PLC-IP3R/PKC signaling by connexin hemichannel-mediated ATP release in rat ventricular myocytes. Possible link between this signaling and NOX/NOS upon shear-mediated spark enhancement in ventricular myocytes needs further investigation. 2661-Pos Board B268 Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease Megan R. McCarthy, Robyn T. Rebeck, Razvan L. Cornea, David D. Thomas. Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. We have used time-resolved fluorescence (FRET) and EPR (DEER) spectroscopies to study the structural changes in calmodulin (CaM) that are relevant to regulation of the muscle calcium release channel, the ryanodine receptor (RyR). Regulation of RyR by CaM is disrupted by oxidation and diseasecausing mutations. However, the structural basis for these regulatory changes, and the role CaM plays in the development of heart failure and arrhythmias, is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the correlation between structure and function in physiologically relevant conditions is largely unknown. To test the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, we use site-directed spectroscopy to determine the structural changes that contribute to calcium regulation in skeletal and cardiac muscle. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes (N and C), then attach
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Wednesday, February 15, 2017
spectroscopic probes to those sites. Ryanodine binding measurements are performed to ensure functional integrity of labeled CaM constructs. Then, we measure changes in intra-CaM distance distributions using time-resolved FRET or DEER. Our previous studies employed DEER of isolated CaM. Now, we rely on time-resolved FRET, to resolve structural changes of CaM bound to functional RyR in sarcoplasmic reticulum membranes. We will compare these measurements with those of CaM bound to a peptide corresponding to the CaM-binding domain of RyR (RyR1 residues: 3614-3643). This work was supported by NIH grants AG26160 and HL092097 (to DDT) and a Predoctoral Fellowship from the American Heart Association 15PRE25700131 (to MRM).
pipette þ 30-min pre-incubation of the cells in AIP2 prior to the experiment) also abolished the effect of flecainide (6 mM, both in patch pipette and extracellular solution) on spontaneous Ca waves in voltage-clamp experiments (vehicle: 6.651.2 waves/45s.; flecainide: 2.150.4 waves/45s.; flecainideþ AIP2: 8.151.9 waves/45s.; p<0.05 for veh. vs. flec. and flec.þAIP2 vs. flec.). Importantly, in the absence of flecainide, AIP2 had no significant effect on the frequency of Ca waves compared to vehicle group (5.051.7 waves/45s. vs. 6.651.2 waves/45s., respectively). Our data identifies CaMKII activity as a critical determinant for inhibitory effect of flecainide on arrhythmogenic Ca waves in Casq2 -/- ventricular myocytes.
2662-Pos Board B269 Effects of Expression Levels of WT and Mutant RyR2 on Ca2D Homeostasis in HEK Cells Nagomi Kurebayashi1, Takashi Murayama1, Naoyuki Tetsuo1, Ryosaku Ohta2, Fumiyoshi Yamashita2, Takashi Sakurai1. 1 Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan, 2Department of Drug Delivery Research, Kyoto University, Kyoto, Japan. Background: Type 2 ryanodine receptor (RyR2) is the Ca2þ release channel on sarcoplasmic reticulum and plays a pivotal role in cardiac E-C coupling. The mutations in RyR2 have been implicated in various arrhythmogenic disorders including catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation (IVF) and long QT syndrome (LQTS). We have characterized their Ca2þ release properties using a heterologous HEK293 expression system and found that CPVT mutants are all gain-offunction (GOF) mutations whereas IVF and LQTS mutants include two groups, GOF and loss-of-function (LOF) types. Among them, the GOF mutations exhibit reduced ER Ca2þ level, depending on the Ca2þ release activity. However, ER Ca2þ level of CPVT patients may not be so reduced because neither structural abnormality nor contractile dysfunction is detected in them. This suggest that expression level of RyR2 in HEK cells may be very high and reduce ER Ca2þ level. In this study we examined effects of expression level of RyR2 on Ca2þ homeostasis in HEK293 cells. Methods: Expression of WT and mutant RyR2s were induced by doxycycline. Cytoplasmic Ca2þ ([Ca2þ]cyt) and ER Ca2þ ([Ca2þ]ER) were determined with fluo-4 and R-CEPIA1er, respectively. Ca2þ induced Ca2þ release (CICR) activity of WT and mutant RyR2s was determined by [3H]ryanodine binding assay. Results: WT and mutant RyR2 protein similarly increased with time after induction and reached steady state at ~24 h. WT and GOF mutants showed spontaneous periodic increase in [Ca2þ]cyt (Ca2þ oscillation) and corresponding decrease in [Ca2þ]ER, and the oscillation frequency was higher in GOF mutants than in WT. The upper level of the [Ca2þ]ER (threshold [Ca2þ]ER) gradually decreased with time after induction and reached steady state at 24 h. There was a very good correlation between threshold [Ca2þ]ER at 24 h and the CICR activity. On the other hand, LOF mutants showed no oscillations with constant [Ca2þ]ER at any time after induction. At 2-4 h after induction, threshold [Ca2þ]ER was similar between WT, GOF and LOF mutants, whereas only GOF, but not WT or LOF mutants, showed Ca2þ oscillations. Discussion: Our results indicate that [Ca2þ]ER in HEK expression system is determined by both CICR activity and expression level of RyR2, and that the [Ca2þ]ER at ~24 h is a sensitive indicator of CICR activity of RyR2 mutants. Implication of LOF mutants in Ca2þ homeostasis will be also discussed based on the expression level.
2664-Pos Board B271 Cardiac Specific IP3R Over-Expression: IP3ICR Contribution in Ca2D Signaling Joaquim Blanch i Salvador, Marcel Wullschleger, Marcel Egger. Physiology, University of Bern, Bern, Switzerland. Inositol-1,4,5-trisphosphate (IP3) is a second messenger produced upon agonist binding to a G-protein coupled receptor (GPCR) and subsequently triggers SRCa2þ release through openings of IP3 receptors (IP3Rs). In cardiac muscle, IP3R type 2 (IP3R2) is the predominant isoform expressed both in ventricle and atrial tissue. Several studies have focused on the functional interaction between ryanodine receptors (RyRs) and IP3Rs in atrial myocytes. However, it is still unclear how IP3-induced Ca2þ release (IP3ICR) may contribute to excitation-contraction coupling in ventricle. Evidence suggests that IP3ICR modulates the RyR function by affecting its local Ca2þ environment. Under pathophysiological cellular remodeling conditions (e.g. atrial fibrillation and heart failure) a functional interplay of IP3R and RyR Ca2þ events may be significantly pronounced. Our aim in this study was to examine this interaction in a cardiac-specific IP3R2 overexpressing (TG) mouse model. Our experimental approach includes: characterization of the IP3 signaling pathway by various pharmacological interventions; electrophysiology under whole-cell configuration of the patch clamp technique in combination with rapid confocal Ca2þ imaging and complemented with molecular biology approaches (RTPCR, Western Blot, immunostaining). Western blot results show an increase in IP3R2 protein expression in the TG model both in atria and ventricular tissue compared to its wild-type littermate (FVB). However, a similar distribution on the junctional SR was found, where IP3Rs co-localize with RyRs. Supporting the protein data, preliminary results show an increase in basal spark frequency (SpF) in TG atrial and ventricular myocytes compared to FVB. Upon IP3 stimulation FVB atrial and ventricular cardiomyocytes presented an increase in SpF. Furthermore, a larger increment was seen in TG atrial myocytes. In order to examine the contribution of the SR-Ca2þ leak mediated by IP3R2 a SR-Ca2þ leak protocol was established. However, even under control conditions a significant contribution of IP3ICR on the SR-Ca2þ leak was found. We conclude that overexpression of IP3R2 in the TG model affects Ca2þ handling significantly by functional interplay with the RyR2.
2663-Pos Board B270 Effective Suppression of Arrhythmogenic Ca Waves by Flecainide in Ventricular Myocytes from Casq2 -/- Mice Depends on CaMKII Activity Dmytro Kryshtal, Nieves Gomez-Hurtado, Bjorn Knollmann. Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA. We reported previously that flecainide effectively suppresses spontaneous Ca waves in both membrane-permeabilized and freshly isolated intact ventricular myocytes from Casq2-/- mice. We also provided strong evidences that this effect is due to the interaction between flecainide and RyR2 and is unrelated to flecainide’s block of Na channels. Moreover, this wave-suppressing effect is dependent on the state of RyR2 (i.e. flecainide is much more potent in Casq2 -/- myocytes, where RyR2 activity is elevated comparing to WT). Here we demonstrate that the effect of flecainide on Ca waves is determined by the activity of CaMKII. In membrane-permeabilized Casq2 -/- ventricular myocytes, the IC50 for flecainide (20-25 min. incubation) was reduced form 6 mM to 1 mM by the presence of 100 nM free CaM. Application of selective CaMKII blockers AIP2 (1 mM) or KN-93 (10 mM) abolished the suppressive effect of flecainide (10 mM) on the incidence, frequency and amplitude of spontaneous Ca waves both in the presence and absence of 100 nM free CaM. In freshly-isolated intact cardiomyocytes, application of AIP2 (1 mM in patch
2665-Pos Board B272 Dynamical Interactions of Early Afterdepolarizations with Stretch Activated Channels Dexter K. Luu, Daisuke Sato, Yuanfang Xie. University of California, Davis, Davis, CA, USA. Cardiac arrhythmia is often triggered by premature ventricular contractions (PVCs), which have been linked to early and delayed afterdepolarizations (EADs / DADs). The cardiac action potential controls mechanical activity of the heart via excitation-contraction coupling. Conversely, mechanical contraction also affects the action potential via stretch-activated channels (SACs). This is called mechano-electric feedback. However, the role of mechano-electric feedback in the formation of afterdepolarizations is not well-understood. In this study, we investigate how activation of SACs promotes or suppress EADs using a physiologically detailed computational model of a rabbit ventricular myocyte. The SAC current (ISAC) is modelled based on Galice et al (BJ 2016). We found that depending on the reversal potential of ISAC, the maximum conductance of ISAC, and the amount of stretch, ISAC can promote or suppress EADs. Mathematical analysis reveals the dynamical origin of these EADs. These results may shed light on the role of SACs on cardiac arrhythmias. 2666-Pos Board B273 Modelling Calcium-Induced-Calcium-Release from Measurements of RyR Gating Derek R. Laver1, Cherrie H. Kong2, Mark B. Cannell2. 1 Biomedical Sciences, University of Newcastle, Callaghan, Australia, 2 Department of Physiology, Pharma cology and Neuroscience, University of Bristol, Bristol, United Kingdom. Many cells depend on ‘calcium-induced calcium release’ (CICR), an inherently regenerative process due to the Ca2þ-dependent gating and Ca2þ conduction of
539a
physiological and pathological conditions. We used fluorescence imaging to measure mitochondrial membrane potential and intracellular calcium concentration ([Ca2þ]i) in primary cultures of hippocampal or cortical neurons (10-15 days in vivo), using Rhodamine123 and Fura-ff respectively. Application of glutamate (50-100mM) causes a stereotypical response consisting of an initial transient spike in [Ca2þ]i followed by a secondary increase in [Ca2þ]i which is coincident with a delayed mitochondrial collapse. Pretreatment of the neurons and astrocytes with different length of polyP significantly reduced the number of cells with a secondary delayed calcium deregulation induced by high concentration of glutamate. Moreover, glutamateinduced mitochondrial depolarization was also prevented by PolyP addition. Importantly, long chain PolyP successfully protected cells against glutamate induced cell death. Thus, polyP protects neurons against glutamate-induced excitotoxicity by reduction of the calcium overload and restoring mitochondrial function.
Intercellular Calcium Channels and Calcium Sparks and Waves II 2660-Pos Board B267 ATP Release through Gap Junction Hemichannels Increases Ca2D Spark Occurrence via P2Y Purinoceptor Signaling in Rat Ventricular Myocytes under Shear Stress Jun Wang, Joon-Chul Kim, Sun-Hee Woo. College of Pharmacy, Chungnam National University, Daejeon, Korea, Republic of. Shear stress in ventricles increases under pressure/volume overload caused by valve diseases, heart failure, and hypertension and enhances Ca2þ transients in ventricular myocytes. We have previously proposed that the shear-mediated Ca2þ transient increase is in part due to sensitization of Ca2þ release sites through ROS generation by NADPH oxidase (NOX) and NO synthase (NOS). Here, we investigated remaining mechanism for the activation of Ca2þ release sites under shear stress in rat ventricular myocytes. Shear stress of ~16 dyn/cm2 was applied onto single cells using micro-jet apparatus. Two-dimensional confocal Ca2þ imaging was performed at 30 Hz. The frequency of resting Ca2þ sparks was immediately increased to ~180% and further increased to ~250% by prolonged exposure (20 s). Pretreatment of cells with inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor 2-APB (2 mM) or protein kinase C (PKC) inhibitor chelerythrine (2 mM) partly (about 50%) suppressed both immediate and prolonged shear effects on the spark occurrence. Blockade of P2 purinoceptors (30 mM suramin) almost completely suppressed shear-induced spark enhancements. Inhibition of phospholipase C (PLC) using U73122 (5 mM) significantly suppressed (~80%) shear-induced Ca2þ spark increases. Pretreatment of P2Y1 receptor antagonist MRS2179 (400 nM) diminished immediate and late increases in spark occurrence during shear stimulation by 60-70%. Inhibition of gap junction hemichannels using carbenoxolone (50 mM) or external treatment of ATP metabolizing apyrase (2 units/ml) eliminated the stimulatory effects of shear stress on the spark occurrence. Consistently, luciferin-luciferase assay revealed ATP release from these myocytes by shear stress within 2 s. These results suggest that shear stress may enhance Ca2þ spark occurrence partly via activation of P2Y-PLC-IP3R/PKC signaling by connexin hemichannel-mediated ATP release in rat ventricular myocytes. Possible link between this signaling and NOX/NOS upon shear-mediated spark enhancement in ventricular myocytes needs further investigation. 2661-Pos Board B268 Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease Megan R. McCarthy, Robyn T. Rebeck, Razvan L. Cornea, David D. Thomas. Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. We have used time-resolved fluorescence (FRET) and EPR (DEER) spectroscopies to study the structural changes in calmodulin (CaM) that are relevant to regulation of the muscle calcium release channel, the ryanodine receptor (RyR). Regulation of RyR by CaM is disrupted by oxidation and diseasecausing mutations. However, the structural basis for these regulatory changes, and the role CaM plays in the development of heart failure and arrhythmias, is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the correlation between structure and function in physiologically relevant conditions is largely unknown. To test the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, we use site-directed spectroscopy to determine the structural changes that contribute to calcium regulation in skeletal and cardiac muscle. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes (N and C), then attach
540a
Wednesday, February 15, 2017
spectroscopic probes to those sites. Ryanodine binding measurements are performed to ensure functional integrity of labeled CaM constructs. Then, we measure changes in intra-CaM distance distributions using time-resolved FRET or DEER. Our previous studies employed DEER of isolated CaM. Now, we rely on time-resolved FRET, to resolve structural changes of CaM bound to functional RyR in sarcoplasmic reticulum membranes. We will compare these measurements with those of CaM bound to a peptide corresponding to the CaM-binding domain of RyR (RyR1 residues: 3614-3643). This work was supported by NIH grants AG26160 and HL092097 (to DDT) and a Predoctoral Fellowship from the American Heart Association 15PRE25700131 (to MRM).
pipette þ 30-min pre-incubation of the cells in AIP2 prior to the experiment) also abolished the effect of flecainide (6 mM, both in patch pipette and extracellular solution) on spontaneous Ca waves in voltage-clamp experiments (vehicle: 6.651.2 waves/45s.; flecainide: 2.150.4 waves/45s.; flecainideþ AIP2: 8.151.9 waves/45s.; p<0.05 for veh. vs. flec. and flec.þAIP2 vs. flec.). Importantly, in the absence of flecainide, AIP2 had no significant effect on the frequency of Ca waves compared to vehicle group (5.051.7 waves/45s. vs. 6.651.2 waves/45s., respectively). Our data identifies CaMKII activity as a critical determinant for inhibitory effect of flecainide on arrhythmogenic Ca waves in Casq2 -/- ventricular myocytes.
2662-Pos Board B269 Effects of Expression Levels of WT and Mutant RyR2 on Ca2D Homeostasis in HEK Cells Nagomi Kurebayashi1, Takashi Murayama1, Naoyuki Tetsuo1, Ryosaku Ohta2, Fumiyoshi Yamashita2, Takashi Sakurai1. 1 Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan, 2Department of Drug Delivery Research, Kyoto University, Kyoto, Japan. Background: Type 2 ryanodine receptor (RyR2) is the Ca2þ release channel on sarcoplasmic reticulum and plays a pivotal role in cardiac E-C coupling. The mutations in RyR2 have been implicated in various arrhythmogenic disorders including catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation (IVF) and long QT syndrome (LQTS). We have characterized their Ca2þ release properties using a heterologous HEK293 expression system and found that CPVT mutants are all gain-offunction (GOF) mutations whereas IVF and LQTS mutants include two groups, GOF and loss-of-function (LOF) types. Among them, the GOF mutations exhibit reduced ER Ca2þ level, depending on the Ca2þ release activity. However, ER Ca2þ level of CPVT patients may not be so reduced because neither structural abnormality nor contractile dysfunction is detected in them. This suggest that expression level of RyR2 in HEK cells may be very high and reduce ER Ca2þ level. In this study we examined effects of expression level of RyR2 on Ca2þ homeostasis in HEK293 cells. Methods: Expression of WT and mutant RyR2s were induced by doxycycline. Cytoplasmic Ca2þ ([Ca2þ]cyt) and ER Ca2þ ([Ca2þ]ER) were determined with fluo-4 and R-CEPIA1er, respectively. Ca2þ induced Ca2þ release (CICR) activity of WT and mutant RyR2s was determined by [3H]ryanodine binding assay. Results: WT and mutant RyR2 protein similarly increased with time after induction and reached steady state at ~24 h. WT and GOF mutants showed spontaneous periodic increase in [Ca2þ]cyt (Ca2þ oscillation) and corresponding decrease in [Ca2þ]ER, and the oscillation frequency was higher in GOF mutants than in WT. The upper level of the [Ca2þ]ER (threshold [Ca2þ]ER) gradually decreased with time after induction and reached steady state at 24 h. There was a very good correlation between threshold [Ca2þ]ER at 24 h and the CICR activity. On the other hand, LOF mutants showed no oscillations with constant [Ca2þ]ER at any time after induction. At 2-4 h after induction, threshold [Ca2þ]ER was similar between WT, GOF and LOF mutants, whereas only GOF, but not WT or LOF mutants, showed Ca2þ oscillations. Discussion: Our results indicate that [Ca2þ]ER in HEK expression system is determined by both CICR activity and expression level of RyR2, and that the [Ca2þ]ER at ~24 h is a sensitive indicator of CICR activity of RyR2 mutants. Implication of LOF mutants in Ca2þ homeostasis will be also discussed based on the expression level.
2664-Pos Board B271 Cardiac Specific IP3R Over-Expression: IP3ICR Contribution in Ca2D Signaling Joaquim Blanch i Salvador, Marcel Wullschleger, Marcel Egger. Physiology, University of Bern, Bern, Switzerland. Inositol-1,4,5-trisphosphate (IP3) is a second messenger produced upon agonist binding to a G-protein coupled receptor (GPCR) and subsequently triggers SRCa2þ release through openings of IP3 receptors (IP3Rs). In cardiac muscle, IP3R type 2 (IP3R2) is the predominant isoform expressed both in ventricle and atrial tissue. Several studies have focused on the functional interaction between ryanodine receptors (RyRs) and IP3Rs in atrial myocytes. However, it is still unclear how IP3-induced Ca2þ release (IP3ICR) may contribute to excitation-contraction coupling in ventricle. Evidence suggests that IP3ICR modulates the RyR function by affecting its local Ca2þ environment. Under pathophysiological cellular remodeling conditions (e.g. atrial fibrillation and heart failure) a functional interplay of IP3R and RyR Ca2þ events may be significantly pronounced. Our aim in this study was to examine this interaction in a cardiac-specific IP3R2 overexpressing (TG) mouse model. Our experimental approach includes: characterization of the IP3 signaling pathway by various pharmacological interventions; electrophysiology under whole-cell configuration of the patch clamp technique in combination with rapid confocal Ca2þ imaging and complemented with molecular biology approaches (RTPCR, Western Blot, immunostaining). Western blot results show an increase in IP3R2 protein expression in the TG model both in atria and ventricular tissue compared to its wild-type littermate (FVB). However, a similar distribution on the junctional SR was found, where IP3Rs co-localize with RyRs. Supporting the protein data, preliminary results show an increase in basal spark frequency (SpF) in TG atrial and ventricular myocytes compared to FVB. Upon IP3 stimulation FVB atrial and ventricular cardiomyocytes presented an increase in SpF. Furthermore, a larger increment was seen in TG atrial myocytes. In order to examine the contribution of the SR-Ca2þ leak mediated by IP3R2 a SR-Ca2þ leak protocol was established. However, even under control conditions a significant contribution of IP3ICR on the SR-Ca2þ leak was found. We conclude that overexpression of IP3R2 in the TG model affects Ca2þ handling significantly by functional interplay with the RyR2.
2663-Pos Board B270 Effective Suppression of Arrhythmogenic Ca Waves by Flecainide in Ventricular Myocytes from Casq2 -/- Mice Depends on CaMKII Activity Dmytro Kryshtal, Nieves Gomez-Hurtado, Bjorn Knollmann. Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA. We reported previously that flecainide effectively suppresses spontaneous Ca waves in both membrane-permeabilized and freshly isolated intact ventricular myocytes from Casq2-/- mice. We also provided strong evidences that this effect is due to the interaction between flecainide and RyR2 and is unrelated to flecainide’s block of Na channels. Moreover, this wave-suppressing effect is dependent on the state of RyR2 (i.e. flecainide is much more potent in Casq2 -/- myocytes, where RyR2 activity is elevated comparing to WT). Here we demonstrate that the effect of flecainide on Ca waves is determined by the activity of CaMKII. In membrane-permeabilized Casq2 -/- ventricular myocytes, the IC50 for flecainide (20-25 min. incubation) was reduced form 6 mM to 1 mM by the presence of 100 nM free CaM. Application of selective CaMKII blockers AIP2 (1 mM) or KN-93 (10 mM) abolished the suppressive effect of flecainide (10 mM) on the incidence, frequency and amplitude of spontaneous Ca waves both in the presence and absence of 100 nM free CaM. In freshly-isolated intact cardiomyocytes, application of AIP2 (1 mM in patch
2665-Pos Board B272 Dynamical Interactions of Early Afterdepolarizations with Stretch Activated Channels Dexter K. Luu, Daisuke Sato, Yuanfang Xie. University of California, Davis, Davis, CA, USA. Cardiac arrhythmia is often triggered by premature ventricular contractions (PVCs), which have been linked to early and delayed afterdepolarizations (EADs / DADs). The cardiac action potential controls mechanical activity of the heart via excitation-contraction coupling. Conversely, mechanical contraction also affects the action potential via stretch-activated channels (SACs). This is called mechano-electric feedback. However, the role of mechano-electric feedback in the formation of afterdepolarizations is not well-understood. In this study, we investigate how activation of SACs promotes or suppress EADs using a physiologically detailed computational model of a rabbit ventricular myocyte. The SAC current (ISAC) is modelled based on Galice et al (BJ 2016). We found that depending on the reversal potential of ISAC, the maximum conductance of ISAC, and the amount of stretch, ISAC can promote or suppress EADs. Mathematical analysis reveals the dynamical origin of these EADs. These results may shed light on the role of SACs on cardiac arrhythmias. 2666-Pos Board B273 Modelling Calcium-Induced-Calcium-Release from Measurements of RyR Gating Derek R. Laver1, Cherrie H. Kong2, Mark B. Cannell2. 1 Biomedical Sciences, University of Newcastle, Callaghan, Australia, 2 Department of Physiology, Pharma cology and Neuroscience, University of Bristol, Bristol, United Kingdom. Many cells depend on ‘calcium-induced calcium release’ (CICR), an inherently regenerative process due to the Ca2þ-dependent gating and Ca2þ conduction of