UNDERSTANDING THE ROLES OF NCX-NAK COUPLING USING COMPUTATIONAL TOOLS by Lulu Chu A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland September, 2016 Abstract Normal cardiac excitability depends on the coordinated activity of ion channels and transporters. Mutations or dysregulation in ion channels affecting their biophysical properties have been known for years as a root cause of fatal human electrical rhythm disturbance (arrhythmias). Moreover, recent studies have shown that defects in ion channel associated protein, ankryin-B, results in a great loss of sodium (Na+)/calcium (Ca2+) exchanger (NCX1) and sodium (Na+)/potassium (K+) ATPase (NaK) to the t- tubules and cause arrhythmia with beta-adrenergic stimulation. It is important to gain a good understanding of their modulation in ionic homeostasis and Ca2+ dynamics in normal and pathological cardiac functions. A biophysically constrained computational model of cardiac ventricular myocyte provides the framework for investigating the functional coupling of NCX1 and NaK and for identifying the underlying mechanisms for early-afterdepolarization generation in cardiomyocytes. The cardiac NCX1 is an electrogenic membrane transporter that regulates Ca2+ homeostasis in cardiomyocytes, serving mainly to extrude Ca2+ during diastole. The direction of Ca2+ transport reverses at membrane potentials near that of the action potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of NCX1 in cardiac Ca2+-induced Ca2+ release (CICR). Interest has been reinvigorated by a recent super-resolution optical imaging study suggesting that ~18% of NCX1 co-localize with ryanodine receptor (RyR2) clusters, and ~30% of additional NCX1 are localized to within ~120 nm of the nearest RyR2. NCX1 may therefore occupy a privileged position in which to modulate CICR. To examine this question, we have developed a mechanistic biophysically detailed model of NCX1 that describes both NCX1 transport kinetics and ii Ca2+-dependent allosteric regulation. This NCX1 model was incorporated into a previously developed super-resolution model of the Ca2+ spark as well as a computational model of the cardiac ventricular myocyte that includes a detailed description of CICR with stochastic gating of L-type Ca2+ channels and RyR2s, and that accounts for local Ca2+ gradients near the dyad via inclusion of a peri-dyadic (PD) compartment. Both models predict that increasing the fraction of NCX1 in the dyad and PD decreases spark frequency, fidelity, and diastolic Ca2+ levels. On the other hand, there is mounting evidence suggesting that there exists a Na+ fuzzy space that is regulated by NCX1, NaK, and neuronal Na+ channels. The model constraints suggest a similar distribution between NaK and NCX1. Furthermore, upon voltage activation, neuronal Na+ channels can raise + the local [Na ]d to ~35-40mM and reverse NCX1 to enhance the CICR process. Therefore, NCX1 plays an important role in promoting Ca2+ entry into the dyad, and hence contributing to the trigger for RyR2 release at depolarized membrane potentials and in the presence of elevated local Na+ concentration. In Ankyrin-B defect cells, pro- arrhythmic spontaneous release and afterdepolarizations are observed in the presence of beta-adrenergic stimulation. Model simulations demonstrates that local regulation of [Ca2+] and [Na+] is compromised as a result of NCX1 and NaK reduction, which contribute to an elevation in Ca2+ spark activities and increased RyR2 opening probability, which leads to further activation of NCX1 and results in imbalance of currents. This underlies the early-afterdepolarization generation. iii Thesis Committee Members Raimond L. Winslow, The Raj and Neera Singh Professor, Department of Biomedical Engineering, The Johns Hopkins University Brian O’Rourke, Vice Chair of Basic and Translational Research, Department of Medicine, The Johns Hopkins University Gordon Tomaselli, Chief, Department of Medicine, Division of Cardiology, The Johns Hopkins University Joseph L. Greenstein, Associate research scientist, Department of Biomedical Engineering, The Johns Hopkins University iv Acknowledgements There are so many people who without which, this dissertation could not have been completed. I really appreciate all the help and guidance that was provided to me over the past 7 years, a really important time period of my life. First and foremost, I must thank my advisor, Dr. Rai Winslow. I could not have asked for a better mentor. You not only taught me about science, research, scientific communication (cardiac electrophysiology, computational models of cardiomyocytes, etc.) but you have been a great role model and have shown me your persistence and passion for science. I have always enjoyed our meetings and discussion and thank you for your tremendous support through my most frustrated time during the experimental work and your help to push me in the right directions. Moreover, I have enjoyed our talks about continuing efforts and persistence in physical activities as well. You constantly inspire me in every single way. To Joe Greenstein, I want to thank you for your patience and all your help with my Ph.D. studies here. You are always so calm and encouraging whenever I am stressed or frustrated with research. I have learned a lot from you from science, research to life attitude. I must also express my gratitude to my committee, Dr. Brian O’Rourke and Dr. Gordon Tomaselli, for their encouragement, guidance and review of my work. To the rest of the Winslow and Mac Gabhann labs, I want to thank you all being a great resource of both knowledge and encouragement. Yasmin, Laura, Rob, Pegy, Mark, Bhaskar, Iraj, Lindsay, Feilim, Liz, An-Chi, Claire, it has been really great working with v you all. In addition, I was thankful for the discussions I had with Dr. Jon Lederer and Dr. G.S. Blair Williams for the early-stage discussion during my NCX1 project. To Kyle Reynolds, I would love to thank you for your help with cluster and any other computer matters that happened over the years. And to my family, in particular my parents, I want to thank you so much for your love and support over the years. Even though you are far away, you have always encouraged me to keep going and overcome any obstacles that come in my way. You are always there for me whenever I need someone to talk to during challenging times. All of the sacrafices you have made through the years to provide me with the best education have not gone unnoticed. Words cannot describe my gratitude for your constant love. vi Table of Contents Chapter 1 - Introduction .................................................................................................. 1 1.1 Objective ............................................................................................................................... 1 1.2 Cardiac Action Potential ..................................................................................................... 1 1.3 Excitation-Contraction Coupling ....................................................................................... 2 1.4 Cardiac dyad ........................................................................................................................ 3 1.5 Sodium Calcium Exchanger ............................................................................................... 4 1.6 Na+ Fuzzy Space ................................................................................................................... 5 1.7 Ankyrin-B and Localization of NCX1 and NaK ............................................................... 6 1.8 Mathematical Modeling of Electrophysiology .................................................................. 8 Chapter 2 - NCX1 regulation of CICR ......................................................................... 13 2.1 Background ........................................................................................................................ 13 2.2 Methods .............................................................................................................................. 16 2.2.1 NCX1 Kinetic Model with CBD12-mediated Allosteric Activation ........................... 16 2.2.2 NCX1 in the Super-resolution Spark Model ................................................................ 25 2.2.3 Spatial Localization of NCX1 in the Canine Whole-cell Model ................................. 27 2.3 Results ................................................................................................................................. 30 2+ 2+ 2.3.1 INCX1 driven by [Ca ]d and [Ca ]i ............................................................................... 31 2+ 2.3.2 Effect of NCX1 on Ca sparks in the SRS model ....................................................... 32 2+ 2.3.3 Role of NCX1 localization on Ca sparks in the whole-cell model ............................ 37 2.3.4 NCX1 localization and the cardiac action potential ..................................................... 39 2.3.5 Does NCX1 play a role in CICR? ................................................................................ 44 2.3.6 Role of NCX1 localization on whole-cell INCX1 ........................................................... 46 2.3.7 Role of NCX1 allosteric regulation on whole-cell physiology .................................... 48 2.4 Discussion ..........................................................................................................................