Neuronal Intracellular Ca2+ and Na+ Dyshomeostasis in the MDX Mouse

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Monday, February 29, 2016

between basal state of LCR period (time from the prior AP-induced Ca2þ transient to the diastolic LCR) and AP cycle length (APCL) (interval between AP-induced Ca2þ transients) and its regulation by PKA-CAMKII dependent phosphorylation in single, isolated rabbit SANC. The role of LCRs in pacemaker activity of guinea-pig SANC or even their presence in the basal state has been recently challenged, but not systematically tested. To probe whether the guinea-pig SANC generate spontaneous diastolic LCRs and their role in the pacemaker firing, LCRs and beating rate in guinea-pig (n=8) were compared to that in rabbit SANC (n=43) at 35.5C. Rhythmically beating guinea-pig SANC do, indeed, generate spontaneous diastolic LCRs beneath sarcolemma, detected via line-scan confocal imaging (fluo-4AM,10uM) when the scanline was positioned beneath sarcolemma parallel to the long axis of the cell. Furthermore, the average LCR characteristics and the and amplitude of the LCR Ensemble Ca2þ signal, the fundamental LCR parameter that ‘‘talks to’’ NCX during diastole were comparable in guinea-pig and rabbit SANC (Table). Relationship between LCR period and APCL were subtended by the same linear function (R2=0.95). That LCRs in both guinea-pig and rabbit SANC contribute to the late diastolic depolarization and regulation of APCL indicate that coupled clock functions involved in pacemaker function are not species limited. Table guinea-pig rabbit LCR amplitude (F/F0)1.3050.04*1.4350.03 LCR size(mm)7.3651.20***4.92 50.15 LCR duration(ms)39.7855.836.8951.51 LCR per cycle(Hz/100mm)6.1051.44**16.2351.51 LCRCa2þ signal(mmxmsxDF/F0)38.3859.9028.3053.42 LCR Ensemble Ca2þ signal (product of mmxmsxDF/F0)320.24595.63236.84537.51 LCR period(ms)310.38541.88315.09512.19 APCL(ms)338.02 534.09378.9259.79 1291-Pos Board B268 New Targeted Ca2D Probes Reveal Mitochondrial Ca2D Signaling Plays a Critical Role in Rat Sinoatrial (SA) Nodal Pacing Xiaohua Zhang1, Christine Couch1, Katalin Torok2, Martin Morad1. 1 Cardiac signaling center of USC, MUSC, & Clemson University, Charleston, SC, USA, 2Institute of Cardiovascular and Cell Science, St George’s, University of London, London, United Kingdom. It remains uncertain to what extent cardiac pacing is governed by ‘‘membrane’’ or ‘‘Ca2þ clocks’’ and what in fact is the master oscillator of the heart? As we have already reported that rapid release of mitochondrial Ca2þ is critical in spontaneous beating of neonatal rat cardiomyocytes, we investigated whether a similar mechanism is at play in SA-nodal cells. Adult rat SA-nodal cells were enzymatically isolated and were cultured for 12 hours prior to viral infection with two genetically encoded fluorescent mitochondria-targeted Ca2þ probes GCaMP3, Kd 1 & 3mM. After 2-3 days in culture, the targeted GCaMP3-probes produced the typical punctate mitochondrial pattern in spontaneously beating myocytes. Such cells had robust expression of If, ICa, and INCX. Simultaneously recorded TIRF images of cytosolic (Cai, dialyzed K5Fura-2) and mitochondrial (Cam, GCaMP3 probes) Ca2þ distributions in blebistatin-treated cells showed differential patterns of rise and fall of Calcium. It was noted that only ~50% of spontaneously beating SA-nodal cells expressed If when hyperpolarized negative to 70mV. Under current-clamp conditions, spontaneously triggered action potentials accompanied by in-phase Cai and Cam signals were recorded only in cells dialyzed with low concentrations of Ca2þ buffer (0.1 mM EGTA & Ca2þ). Withdrawal of extracellular Naþ increased the rate of spontaneously generated Cai-oscillations at fixed holding potentials, but triggered diverse signals in different mitochondrial populations that ranged from continuous uptake of Ca2þ to spontaneously triggered oscillations to releasing of Ca2þ. Similarly, application of caffeine during Naþ withdrawal while increasing Cai, showed Cam signals indicative of up-take of Ca2þ at some locations and release at others. We conclude that SR and mitochondrial Ca2þ signals and not If serve as the master oscillator of SA nodal cells. Supported by NIH Ro1HL-15162 1292-Pos Board B269 Preveleged Ca2D Signaling Pathway between Membrane NCX and Mitochondria in Cardiac Myocytes Xiaohua Zhang, Naohiro Yamaguchi, Lars Cleemann, Martin Morad. Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA. Accumulating evidence suggests that mitochondrial Ca2þ signaling plays a critical role in cardiac myocytes E-C coupling and that this may involve

Ca2þ entering the mitochondria via the mitochondrial Ca2þ uniporter (MCU) and exiting on the mitochondrial Naþ-Ca2þ exchanger (mNCX). Here we investigate whether there are privileged Ca2þ transport pathways between surface membrane, mitochondria and SR. To address this issue, we used genetically engineered mitochondrial Ca2þ probes based on GCamP3 and GCamP6 along with synthetic fluorescent dye, Fura-2, to image mitochondrial and cytosolic Ca2þ movements simultaneously. Mitochondrial Ca2þ probes were virally introduced in either adult or neonatal cardiomyocytes. Whole-cell patch clamping and high-resolution TIRF microscopy were used to measure membrane current, mitochondrial Ca2þ and subsarcolemmal cytosolic Ca2þ simultaneously. In whole cell clamped myocytes dialyzed with 2mM EGTA, Naþ withdrawal- (substituting Naþ by tetraethylammonium, TEA) generated an outward NCX-mediated current reflecting Ca2þ entry and caused a rapid increase of mitochondrial Ca2þ, without significant rise of cytosolic Cai, because of Ca2þ-buffering by 2mM EGTA. When Naþ was readmitted, causing a rapid fall (release) of mitochondrial Ca2þ it triggered a significant transient rise of cytosolic Ca2þ despite the presence of buffering by 2mM EGTA. Interestingly, 5mM caffeine puffs while triggering rapid release of Ca2þ from the SR caused little or no significant rise in the mitochondrial Ca2þ signal. Blockers of MCU or mitochondrial NCX failed to suppress this Naþ withdrawal-induced mitochondrial Ca2þ rise. Our results suggest that Ca2þ buffers may block transfer of Ca2þ from the SR via the cytosol to mitochondria without blocking the privileged Ca2þ communication between sarcolemma and mitochondria. Supported by NIH grant R0-1,HL15162 1293-Pos Board B270 Binding Sites of the Ca/Na Exchanger NCX Analyzed with Poisson Fermi Theory Jinn-Liang Liu1, Robert S. Eisenberg2. 1 National Hsinchu University of Education, Hsinchu, Taiwan, 2Rush University, Chicago, IL, USA. The calcium sodium transporter NCX is a Y shaped branched channel in the structure crystallized by Liao et al. (2012) with well-defined binding sites. It seems unlikely that mechanism can be understood until structure is extended by estimates of binding free energies. It seems that existing molecular dynamics cannot cope with trace concentrations and movements of calcium. The number density of ionizable side chains is extremely high and so theory must include finite size ions and large electric and steric free energies. We use Fermi-Poisson theory (Liu and Eisenberg, 2014) that deals successfully with bulk solutions and several channel types. Outputs of theory include electric and steric fields of ions with different sizes; correlations of ions of different charge, and polarization of water, along with number densities of ions, water molecules and interstitial voids. These vary with conditions. They are not assumed. We calculate electrostatic and steric potentials of three Naþ and one Ca2þ binding site of the Liao structure with charges from PDB2PQR software. Atomic and numerical details of binding sites illustrate the ion-exchange mechanism and its energetic endpoints. Energy profiles of Naþ and Ca2þ ions along their pathways give insight into the mechanism of coupling by which NCX moves intracellular Ca2þ uphill against its chemical gradient, while Naþ moves downhill. But the mechanism and energetics of the conformation change of the transporter protein NCX remain a fascinating mystery. NCX does not use energy beyond ion gradients and electric and steric fields described consistently by Poisson-Fermi theory. Its conformation change is that of catalysis, not pumping, in the dictionary sense of ‘pumping’. PoissonFermi can describe energetic end points, although a kinetic model of conformation remains outside its grasp. 1294-Pos Board B271 Neuronal Intracellular Ca2D and NaD Dyshomeostasis in the MDX Mouse Jose R. Lopez1, Juan Kolster2, Jose Adams3. 1 Molecular Biosciences, University of California, Davis, CA, USA, 2Centro de Investigaciones Biome´dicas, Mexico, Mexico, 3Division of Neonatology, Mount Sinai, Medical Center, Miami, FL, USA. Duchenne muscular dystrophy (DMD) is an inherited X-linked disorder characterized by the deficiency of dystrophin as well as intracellular ion (Ca2þ and Naþ) dyshomeostasis in skeletal and cardiac muscles. There is also an absence of dystrophin in cortical neurons of DMD patients and animal models. We hypothesized that similar to muscles, intracellular ion dyshomeostasis may also occur in cortical neurons of mdx mice. We measured resting intracellular [Ca2þ]i and [Naþ]i in primary cortical neurons from 3-month-old mdx mice and age-matched wild type (wt) using Ca2þ- and

Monday, February 29, 2016 Naþ microelectrodes. [Ca2þ]i and [Naþ]i were elevated in mdx neurons compared with wt (by 73% and 50%, respectively). Removal of Ca2þ from bathing media produced a significant reduction of [Ca2þ]i in both genotypes, however the reduction was greater in mdx (45%) versus wt (20%). The stretch ion channel blocker GsMTx-4 (5 mM) significantly decreased [Ca2þ]i and [Naþ]i in both genotypes. [Ca2þ]i was reduced in 38% and 17% in mdx and wt neurons respectively. [Naþ]i was reduced in 28% in mdx and 15% wt neurons. Application of the L-type channel inhibitor nifedipine did not modify [Ca2þ]i in either mdx or wt neurons. However, incubation with 500 mM ryanodine or 10 mM xestospongin C (inositol trisphosphate receptor (IP3R) blocker) partially reduced [Ca2þ]i by 16% and 20%, respectively, in mdx neurons. These results demonstrate that cortical neurons from mdx mice exhibit elevated resting [Ca2þ]i and [Naþ]i as a result, in part, of both abnormal Ca2þ entry through stretch-activated channels and anomalous Ca2þ release via the ryanodine and IP3R. The absence of dystrophin modifies the regulation of [Ca2þ]i and [Naþ]i in mdx neurons and may potentially contribute to altered brain function resulting in cognitive deficits in DMD patients and the mdx mouse model. Supported by Florida Heart Institute. 1295-Pos Board B272 Role of Inorganic Polyphosphate (PolyP) in Physiological and Pathophysiological Response to Glutamate in Mammalian Neurons Plamena R. Angelova, Andrey Y. Abramov. UCL Institute of Neurology, London, United Kingdom. Inorganic polyphosphate (PolyP) is a negatively charged linear polymer that is found in all living organisms where it has multiple roles. Recently, we showed that inorganic polyphosphate can act as a calcium-dependent gliotransmitter via P2Y1 purinergic receptors of the astrocytes. However, upon exogenous PolyP application, additionally to all astrocytes, 3% of the neurons respond with increased cytosolic calcium and therefore PolyP may be acting as well as a neurotransmitter. Using highly specific fluorescent indicator for inorganic polyphosphate (JC-D8) that we recently developed and a lentiviral delivery of a range of different fluorescent probes to target various types of vesicles, we visualized the release of polyphosphate and study possible interactions between these vesicles and other cellular organelles in primary co-culture of cortical neurons and astrocytes. Co-localisation analysis has shown that PolyP is located mostly in putative ATP-containing vesicles, to a lesser extent in lysosomes and mitochondria and not in the glutamate-containing ones. In TIRF microscopy experiments we have been able to show in-depth release of these vesicles following physiological stimulation. In our hands, release of PolyP out of those vesicles could be stimulated by application of calcium ionophores, protonophores, gpn, PolyP and changes in pH. In live cell imaging experiments in neurons, application of extracellular PolyP can significantly reduce the physiological calcium response to glutamate, by slowing down the Ca2þ efflux in primary neurons, and as well, depending on the length, prevents the delayed calcium deregulation. Furthermore this modulation appears to act via P2Y1 receptors. This study highlights the importance of PolyP in modulating synaptic transmission and perhaps presents as an attractive target for therapy in diseases, where neuronal damage is caused by excitotoxicity. Supported by Leverhulme Trust. 1296-Pos Board B273 Cortex Phellodendri Extract Relaxes Mouse Airway Smooth Muscle Weiwei Chen, Qui-Ju Jiang, Meng-Fei Yu, Qing-Hua Liu. Institute for Medical Biology, College of Life Sciences, South Central University for Nationalities, Wuhan, China. Asthma is a common chronic respiratory disease. Excessive airway obstruction is a cardinal symptom, which results from the contraction of airway smooth muscle cells (ASMCs). It has been reported that berberine, an active ingredient of Cortex Phellodendri extract (CPE), can induce ASMC relaxation. However, we found that non-berberine component of CPE (a n-butyl alcohol extract of Cortex Phellodendri, NBAECP) has also a similar relaxant effect observed in tracheal rings and lung slices. NBAECP completely inhibited precontraction induced by high Kþ and ACh in a concentrationdependent manner. The precontraction induced by the former was also inhibited by nifedipine, and which induced by the latter was partially reduced by nifedipine (VDCC inhibitor) and pyrazole 3 (NSCC inhibitor). These results indicate that NBAECP can relax ASMCs via inhibiting VDCC and NSCC and imply that NBAECP might be a potential new drug for asthma therapy. Key words: Cortex Phellodendri; asthma, airway smooth muscle

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1297-Pos Board B274 R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes Elisa Bovo, Aleksey V. Zima, Jody L. Martin, Pieter P. de Tombe. Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA. In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats). 1298-Pos Board B275 Simulations of Subcellular Heterogeneity in Calcium Blink Kinetics Elisa Nunez Acosta, Eva Polakova, Eric A. Sobie. Pharmacology, Mount Sinai Hospital, New York, NY, USA. In cardiac myocytes, calcium (Ca2þ) release from the sarcoplasmic reticulum (SR) depends on local calcium concentrations in both the cytosol and the SR. The local dynamics of SR Ca2þ depletion have been visualized experimentally through the recording of Ca2þ ‘‘blinks.’’ Specifically, it has been shown that blink nadir is relatively reproducible between local Ca2þ release sites whereas blink recovery kinetics are highly variable. We applied a spatiallydetailed, stochastic mathematical model to gain insight into the mechanisms underlying this heterogeneity. Novel features of our model include: (1) calculation of Ca2þ fluxes between locations within the junctional SR (JSR) and dyadic spaces; (2) variable numbers and locations of connections between the JSR and the SR network; (3) variable and irregular clusters of ryanodine receptor (RyR) channels in the JSR membrane; (4) spatially-defined localization of SR Ca2þ pumps (SERCA) in JSR and network SR membranes. Using this model, we investigated how different parameters influenced Ca2þ blink amplitude and kinetics. From these simulations we determined that the number of connections between the JSR and the SR network was the most important parameter controlling Ca2þ blink recovery kinetics. For instance, 80% recovery of pre-release JSR [Ca2þ] decreased from 400 ms with 1 connection to 125 ms with 5 connections. SERCA density also influenced Ca2þ blink recovery, but the quantitative strength of this effect was relatively smaller (126 ms at nominal SERCA versus 209 ms with SERCA reduced by 80%). The remaining model parameters had only minor effects on blink kinetics. These results indicate the importance of SR Ca2þ diffusion in release kinetics and suggest mechanisms underlying Ca2þ blink heterogeneity. 1299-Pos Board B276 Dyadic Remodeling as a Cause of Impaired E-C Coupling in Early Stages of Myocardial Injury Alexandra Zahradnikova, Marta Novotova, Katarina Mackova, Ivan Zahradnik, Alexandra Zahradnikova. Department for Muscle Cell Research, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia. In failing myocytes, impaired E-C coupling is often associated with loss of t-tubules and appearance of orphaned calcium release units. Impairment of