Feb 13, 2017 - DHBP was also less effective on heart and skeletal muscle RyR's recon- ... test the hypothesis that hMSC-mediated PS enhances cardiac contractility and .... Allosteric ligands modulate protein activity by altering the energy ...
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Monday, February 13, 2017
in intact single fibers enzymatically isolated from murine flexor digitorum brevis (FDB) muscles. DHBP (10-50 mM) also inhibited SR Ca2þ release in ventricular epicardium of intact hearts. DHBP effects peaked after a few minutes of superfusion and dissipated within minutes upon washout. Unexpectedly, DHBP was a weak inhibitor of RyR-mediated Ca2þ release from skeletal SR microsomes. DHBP was also less effective on heart and skeletal muscle RyR’s reconstituted into planar bilayers, where the drug did not affect open probability and required high doses (~1 mM) to reduce open channel conductance by 50% (which suggest flicker block of the open channel). Our preliminary results suggest that DHBP is an attractive water-soluble, cell-permeable and reversible experimental probe to understand the role of RyR’s in cellular processes. The drug action seems relatively specific (DHBP was also without effects on SERCA-mediated SR Ca2þ loading or ATPase activity). Also, in skeletal muscle DHBP may be an alternative to inhibitors of myosin (BTS, blebbistatin) to arrest muscle contraction. Further research is still required to fully understand DHBP mechanism of action. The larger cellular effects of DHBP (an organic divalent cation) versus isolated RyRs may suggest drug accumulation in the cytosol at very high levels. However, DHBP synergized the effects of imperatoxin and peptide blockers on RyRs reconstituted into bilayers. These results may indicate that DHBP inhibits RyR’s in cells by acting on ancillary proteins, which may dissociate from the channels during the process of cell subfractionation and/or isolation of SR microsomes. 797-Plat Optical Stimulation of iPS Cardiomyocytes allows Brand New Insights into Contractility and Electropyhsiology Conjunctions Sonja Stoelzle-Feix1,2, Matthias Beckler1,2, Patrick Mumm1,2, Ulrich Thomas1,2, Leo Doerr1,2, Elena Dragicevic1,2, Krisztina Juhasz1,2, Corina T. Bot3, Michael George1,2, Andrea Br€uggemann1,2, Niels Fertig1,2, Jean-Francois Rolland2,4, R. Rizzetto2,4, L. Redaelli2,4, Philipp Sasse2,5. 1 Nanion Technologies Gmbh, Munich, Germany, 2OPTEL Consortium, funded by EuroTransBio Initiative, Germany, 3Nanion Technologies Inc, Livingston, NJ, USA, 4Axxam S.p.A., Milan, Italy, 5Life and Brain Center, Inst. f€ ur Physiologie I, Univ. Bonn, Bonn, Germany. Optical in-vitro platforms will be of particular relevance in the early stages drug discovery processes. We show recordings on impedance and extracellular field potential (EFP)-based devices with induced pluripotent stem cell (iPSC)derived cardiomyocytes as well as automated patch clamp data. Optogenetic stimulation and the recording of electrophysiological and contractile parameters of ChR2 (channelrhodopsin 2) transfected iPS Cor.4U cardiomyocytes were performed in a new assay approach, which allows a parallel investigation of impedance and EFP signals. This allowed a mechanistic understanding of cardiomyocyte cell physiology, which has been investigated over a physiological frequency range (60-180 ppm). Frequency dependent effects on cell physiology with reference compounds such as Ranolazine and Mexiletine will be presented. Furthermore, automated patch clamp investigations in the voltage-and currentclamp mode on blue-light activated ChR2 (channelrhodopsin 2) transfected cells will be presented and discussed in association with impedance/EFP results. 798-Plat Human Mesenchymal Stem Cell Paracrine Signaling Counteracts Heterocellular Coupling Effects on Cardiac Contractility and Arrhythomgenicity Joshua Mayourian1, Timothy J. Cashman1, Bryce V. Johnson2, David M. Sachs1, Deepak A. Kaji3, Eric A. Sobie3, Kevin D. Costa1. 1 Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Department of Medicine, University of Washington Seattle, Seattle, WA, USA, 3Icahn School of Medicine at Mount Sinai, New York, NY, USA. Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. Demonstrated benefits include reduced fibrosis and enhanced contractile function, with predominant mechanisms thought to involve paracrine signaling (PS) and heterocellular coupling (HC) between hMSCs and host myocardium. In this study, we utilized mathematical modeling and three-dimensional human engineered cardiac tissues (hECTs) to test the hypothesis that hMSC-mediated PS enhances cardiac contractility and minimizes arrhythmogenicity, counterbalancing the unfavorable effects of direct HC. Based on published studies, our previous hMSC-cardiomyocyte HC model was modified to incorporate hMSC PS effects on single-cell cardiomyocyte ion channel activity and tissue-level fibrosis. Incorporating an established excitation-contraction model, our simulations of PS-only and combined HCþPS effects of hMSCs on human cardiomyocytes replicated our measurements of contractile function of hECTs under matched experimental hMSCmediated treatments. For example, model simulations and hECTs both demonstrated that the hMSC-mediated effects were most beneficial under PS-
only conditions, where developed force significantly increased by 3.5-fold compared to non-hMSC-supplemented controls during physiologic 1-Hz pacing. Similarly, maximum rates of contraction and relaxation were enhanced by PS-only conditions, and diminished by HC. Counteracting PS and HC effects of hMSCs were also revealed in a vulnerable window (VW) analysis of tissuelevel arrhythmogenicity in simulated cardiac tissue with moderate (21%) and high (40%) diffuse fibrosis; hMSC HCþPS conditions had variable effects on VW dependent on the percent of hMSCs delivered, while PS-only conditions consistently decreased the VW, thus minimizing arrhythmogenicity. Together, these findings support our hypothesis, and suggest identifying key hMSC paracrine signaling factors as an alternative hMSC-based cardiac therapy.
Platform: Voltage-gated K Channels and Mechanisms of Voltage Sensing and Gating II 799-Plat Non-Canonical Interactions between Voltage Sensors and Pore Domain in Shaker KD-Channel Joa˜o Carvalho-de-Souza, Francisco Bezanilla. Biochem and Mol Biology, The University of Chicago, Chicago, IL, USA. W434F mutation in the pore domain (PD) of Shaker Kþ-channels yields nonconductive channels, useful for gating currents studies. Comparisons of Q-Vs recorded with W434F with those recorded with absence of Kþ show virtually indistinguishable data. When a mutation in the S3-S4 linker, L361R, is introduced, the Q-V recorded with W434F, and the curve of Kþ conductance activation by voltage (G-V) are both strongly shifted to more negative voltages, and they unexpectedly cross each other. This effect is typical when the channel has more than one open state, which does not seem to be the case according to our single channels recordings. As expected, Q-V curve recorded in the conductive Shaker containing L361R mutation does not cross its G-V. This unprecedentedly showed that W434F-containing PD can potentially affect mutant VSDs although it is virtually silent for WT VSDs. To investigate this noncanonical coupling between VSD and PD, other than via S4-S5 linker, we produced dimers of Shaker that would show a mutant VSD (L361R) CLOSE, or FAR from a mutant PD (W434F). According to the Kv1.2 crystal structure, VSDs are near the PD from the neighbor subunit and we consider that would happen similarly in dimers of shaker. VSD mutations shift the slowinactivation curves to more negative voltages, with the effect being more intense in CLOSE compared to FAR channels. Furthermore, current peaks progressively increase at þ60 mV after 100-ms pre-pulses from 180 to 100 mV differently on CLOSE and FAR channels, indicating the VSD mutation interfere with the PD inactivation according to its relative position. Our data show that VSD and PD are in close communication beyond what is predicted by the S3-S4 connection, especially when mutations are present in both domains of the protein. Support: NIH-GM030376. 800-Plat Voltage Sensing in Hyperpolarization Activated Cyclic Nucleotide Gated (HCN) Channels Karen M. Callahan, Nazzareno D’Avanzo. Pharmacology et Physiologie, Universite´ de Montre´al, Montre´al, QC, Canada. The voltage-sensing domains (VSDs) of HCN channels have topologies similar to other voltage-dependent ion channels, including a series of positively charged residues in their S4 helix that are predicted to move with the same directionality as in other channels. However, HCN channels activate very slowly at hyperpolarized potentials. Intriguingly, despite identical S4 and S4S5 linkers between mammalian isoforms, HCN1-4 activate with different voltage-dependencies and gating kinetics. Here we begin to examine the molecular details of VSD movement in HCN channels through molecular dynamics simulations. Although the gating charge of sea urchin HCN (spHCN) channels has been estimated to be very small compared to that of Kv channels, our data indicates this is obtainable by a similar displacement of the S4 helix. We also compare gating charge estimates of human HCN isoforms, which have been experimentally unattainable to date. Lastly, we examine key interactions between isoforms that may underlie differences in the voltage-gating and kinetics. 801-Plat The Tarantula Toxin Guangxitoxin-1E Traps KD Channel Voltage Sensor in a Fully Resting Conformation Drew C. Tilley, Kenneth S. Eum, Jon T. Sack. Physiology and Membrane Biology, University of California Davis, Davis, CA, USA. Allosteric ligands modulate protein activity by altering the energy landscape of conformational change in ligand-protein complexes. Here, we investigate how
