Calcium Homeostasis Modulator (CALHM1/2) in Pulmonary Arterial Hypertension Item Type text; Electronic Thesis Authors Rodriguez, Marisela Citation Rodriguez, Marisela. (2020). Calcium Homeostasis Modulator (CALHM1/2) in Pulmonary Arterial Hypertension (Master's thesis, University of Arizona, Tucson, USA). Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 03:13:25 Link to Item http://hdl.handle.net/10150/648659 CALCIUM HOMEOSTASIS MODULATOR (CALHM1/2) IN PULMONARY ARTERIAL HYPERTENSION By Marisela Rodriguez Copyright © Marisela Rodriguez 2020 A Thesis Submitted to the Faculty of the COLLEGE OF MEDICINE In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN CLINICAL TRANSLATIONAL SCIENCES In the Graduate College THE UNIVERSITY OF ARIZONA 2020 - 2 - Acknowledgements I would like to acknowledge the members of the Yuan Lab throughout my undergraduate years who inspired me to join research, taught me what it means to be a part of the scientific community, and always showed support for a young student as myself. It has led me to pursue a graduate education in this field and continue as a part of the scientific investigatory community. I would like to thank my thesis committee for their advising and support. Dr. Jiyuan Chen (Era) for contributing and expanding this project with me. I would also like to thank my mentor, Professor Jason Yuan for his patience and teachings, never having a direct way of saying things but allowing for the message to be said in parables and applicable to everything in life. Also, for always reminding me that there are three factors to my work- heart, brain, and hands. - 3 - Dedication This work is dedicated to my family and friends for their unconditional support, encouragement, and belief in me; especially my father for always setting standards and my mother for advising on hard work ethic and both reminding me to implement my all into everything I do. - 4 - TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................................. - 6 - LIST OF TABLES ................................................................................................................................... - 7 - LIST OF ABBREVIATIONS AND ACRONYMS: ................................................................................ - 8 - ABSTRACT .......................................................................................................................................... - 11 - SIGNIFICANCE AND INTRODUCTION ........................................................................................... - 15 - PHENOTYPICAL SWITCH AND PAH .............................................................................................. - 27 - ION CHANNELS AND PULMONARY HYPERTENSION ............................................................... - 33 - CALCIUM HOMEOSTASIS MODULATOR ...................................................................................... - 38 - MATERIALS AND METHODS ........................................................................................................... - 47 - Pulmonary Artery Isolation ................................................................................................................... - 47 - Isolation and preparation of rat PASMC ............................................................................................... - 47 - Western Blotting .................................................................................................................................... - 54 - Polymerase Chain Reaction ................................................................................................................... - 60 - Statistical Analysis ................................................................................................................................. - 65 - RESULTS .............................................................................................................................................. - 66 - CONCLUSION ...................................................................................................................................... - 83 - REFERENCES ...................................................................................................................................... - 86 - - 5 - LIST OF FIGURES Figure 1 – Treatment algorithm of pulmonary arterial hypertension ……………………. 21 Figure 2 – Immunoflourescent staining of human SMCs………………………………... 27 Figure 3 – PASMC Phenotypic Plasticity………………………………………………… 29 Figure 4 – Mechanisms and signaling cascade of Ca2+ in PAH………………………...... 34 Figure 5 – Crystal structure images of human CALHM2 dimer…………………………. 36 Figure 6 – Transmembrane topology of CALHM channel………………………………. 38 Figure 7 – CALHM 1-3 mRNA expression level………………………………………... 42 Figure 8 – Freshly isolated PA branches from rat………………………………………... 49 Figure 9 – Primary PASMC Isolation Steps……………………………………………... 50 Figure 10 – Contractile vs proliferative phenotype markers…………………………….. 64 Figure 11 – Upregulated CALHM1 and 2……………………………………………….. 66 Figure 12 – Upregulated AKT/mTOR signaling proteins……………………………….. 68 Figure 13 – CaSR and Notch upregulated in phenotypical transition……………………. 71 Figure 14 – Pulmonary vascular remodeling in chronic hypoxia………………………… 74 Figure 15 – Rapamycin downregulates CALHM1 and 2………………………………… 77 Figure 16 – Upregulated CALHM1 and 2 in MCT………………………………………. 79 Figure 17 – Upregulated CALHM1 in IPAH hPASMCs………………………………… 79 - 6 - LIST OF TABLES Table 1 – Hemodynamic definition of pulmonary hypertension (PH)…………………… 13 Table 2 – Clinical classification of pulmonary hypertension (PH)………………………. 15 Table 3 – Female to male ratios in major PAH registries………………………………… 18 Table 4 – Drugs approved by FDA to treat PAH…………………………………………. 20 Table 5 – Contractile and proliferative SMC markers…………………………………… 26 Table 6 – CALHM 1-3 PCR primer sequences………………………………………….. 43 - 7 - LIST OF ABBREVIATIONS AND ACRONYMS: ACTA2 Smooth muscle cell actin ANG-1 Angiopoietin-1 AVD Apoptotic volume decrease 2+ 2+ [Ca ]cyt Cytosolic free Ca concentration CALHM Calcium homeostasis modulator Came calmodulin CaSR Calcium-sensing receptor CCE Capacitive Ca2+ entry CO Cardiac Output CO2 Carbon dioxide CpcPH Combined pre-and post-capillary PH CREB cAMP response element binding protein CTEPH Chronic thromboembolic pulmonary hypertension DAG Diacylglycerol EC-coupling Excitation-contraction coupling ECM Extracellular matrix ER Endoplasmic reticulum FBS Fetal bovine serum FGFR Fibroblast growth factor receptor GPCR G protein-coupled receptor HIF-1α Hypoxia-inducible factor 1α HIV Human immunodeficiency virus HPV Hypoxic pulmonary vasoconstriction IH Intimal hyperplasia inh. Inhalation IP3 Inositol 1,4,5-triphosphate IpcPH Isolated post-capillary PH i.v. Intravenous injection LVEF Left ventricle ejection fraction - 8 - MAPK Mitogen-activated protein kinase pathway MHC11 Myosin heavy chain-11 MLCK Myosin light chain kinase mPAP Mean pulmonary arterial pressure mmHg Millimeters mercury NFAT Nuclear factor of activated T-cells NO Nitric oxide NYHA-FC New York Heart Association Functional Classification PA Pulmonary artery PASMC Pulmonary artery smooth muscle cell PAP Pulmonary arterial pressure PAH Pulmonary arterial hypertension PAWP Pulmonary arterial wedge pressure PCH Pulmonary capillary hemangiomatosis PDGF Platelet-derived growth factor PGI2 Prostacyclin PH Pulmonary hypertension PM Plasma membrane p.o. Oral administration PVOD Pulmonary vino-occlusive disease PVR Pulmonary vascular resistance REVEAL Registry to Evaluate Early and Long-term PAH disease management RHC Right heart catheterization ROCC Receptor-operated Ca2+ channel ROCE Receptor-operated Ca2+ entry ROS Reactive oxygen species RTK Receptor tyrosine kinase RV Right ventricle SMC Smooth muscle cell SOCC Store-operated Ca2+ channel SOCE Store-operated Ca2+ entry s.q. Subcutaneous injection - 9 - SR Sarcoplasmic reticulum STIM Stromal interaction molecule TKR Tyrosine kinase receptor TRPC transient receptor potential channel VDCC Voltage-dependent Ca2+ channel VSMC Vascular smooth muscle cell WHO-FC World health organization functional classification WSPH World Symposium on Pulmonary Hypertension WU Wood Units - 10 - ABSTRACT Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. The increased pulmonary arterial pressure (PAP) in patients with PAH is mainly generated by increased pulmonary vascular resistance (PVR) (18, 70, 103). Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular wall stiffness are the major causes for the elevated PVR and PAP in patients with PAH. Concentric pulmonary vascular remodeling is among one of the major causes for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP), also one the major causes for increasing afterload of right ventricle (RV) and inducing right heart failure leading to death if untreated (103). Excessive pulmonary artery smooth muscle cell (PASMC) proliferation and inhibited PASMC apoptosis have been implicated