Imaging Mitochondrial Dynamics in the Adult Heart

Thesis submitted by Siavash Beikoghli Kalkhoran BSc (First class Hons.), MSc (Distinction) For the degree of Doctor of Philosophy University College London, UK.

Institute of Cardiovascular Science The Hatter Cardiovascular Institute, University College London, 67 Chenies Mews, London, WC1E 6HX.

October 2017

Declaration

I, Siavash Beikoghli Kalkhoran, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. The assistance and contribution of individuals to the generation of results are acknowledged within the methods sections of each chapter.

2

Dedicated to Soheila & Taher

3 Abstract

Background

Mitochondrial dynamics, the phenomenon which incorporates inter-mitochondrial communication and changes in mitochondrial morphology is central to cellular homeostasis. Although the phenomenon of mitochondrial dynamics has been comprehensively studied under normal and pathological conditions in non-cardiac cells, and more recently in cardiac cell lines, its relevance to adult cardiomyocytes has not been so well-established and is investigated in this thesis.

Methods and Results

Using 2D and 3D electron microscopy, we initially evaluated the morphological features of the 3 different mitochondrial subpopulations (interfibrillar, peri-nuclear, subsarcolemmal) in adult rodent cardiomyocytes, and demonstrated that they are morphologically unique. These morphological characteristics were found to be altered under pathological conditions such as ischaemia or the genetic ablation of mitochondrial fusion proteins “mitofusins”. Using mice expressing the Dendra2 fluorescence probe, we then confirmed that mitochondrial fusion events (“the inter- mitochondrial communication”) occur in live adult cardiomyocytes, and the fusion rates differ according to the mitochondrial subpopulation. We next performed high throughput screening of a small molecule library and identified hydralazine (a drug used to treat hypertension and heart failure) to be a novel modulator of mitochondrial dynamics, acting to inhibit mitochondrial fission and protect against the detrimental effects of acute myocardial ischaemia/reperfusion injury by preserving mitochondrial dynamics.

4 Conclusion

This thesis has demonstrated that 2D and 3D changes in mitochondrial shape features, as well as alterations in inter-mitochondrial communication, are of high relevance to adult rodent cardiomyocytes. Hydralazine-induced cardioprotection in the setting of

IRI demonstrates the significance of distinct aspects of mitochondrial dynamics and reveals the role they play in the normal functioning of adult cardiomyocytes.

5 Acknowledgements

My profound appreciation and thanks go to my supervisors Prof Derek Hausenloy and

Prof Derek Yellon who were with me every step of the way. Prof Hausenloy, it was an honour to be your student and I am highly grateful for your invaluable support, guidance, directions and kindness for supervising me throughout these years and I hope to work with you again in the near future. Prof Yellon, I am so thankful that you kindly let me take this PhD at the Hatter Institute, and I am proud to be supervised by you.

I would like to especially thank my colleagues Dr Sang Bing Ong at Duke-NUS

Medical School as well as Dr Sapna Arjun, Dr Jaime Riquelme Melendez, Dr Andrew

Hall at the Hatter Cardiovascular Institute for their help with technical aspects of my project including experiment design and assay characterisations. I should also mention and thank my other colleagues, Ms Parisa Samangouei, Dr Jose Miguel Vicencio

Bustamante, Dr Niall De Burca and Dr Sean Davidson and Dr David He for all their fruitful discussion and guidance.

I would like to also express my deepest gratitude to technical staff from UCL and collaborators who contributed significantly to the publication of this thesis including experts in electron microscopy, Prof Gerald Dorn (For providing the mitofusin mice from Centre for Pharmacogenomics of Washington University), Dr Ian white and Dr

Jemima Burden (3D serial section electron tomography, from MRC laboratory of molecular and Cell Biology), Dr Peter Munro (3 view, from Institute of

Ophthalmology, UCL), Dr Mark Turmaine (2D EM, from Department of Cell and

Developmental Biology) and Dr Rebecca Poh. (FIBSEM, from Carl Zeiss Pte. Ltd,

Singapore); the HTS team Dr Janos Kriston-Vizi, Dr. Joana Rodrigues Simoes Da

Costa and Robbin Ketteler (HTS, from MRC Laboratory of Molecular and Cell

6 Biology); confocal and Denrda2 related experiments microscopy Michelle Tan Guet

Khim (Department of Clinical Translational Research, Singapore General Hospital),

Miss Kwek Xiu Yi and Mrs Khairunnisa Binte Katwadi, statistical assistance Dr Qiao

Fan, Dr Bibhas Chakraborty (from Centre for Quantitative Medicine, DUKE-NUS) and Miss Jackie Cooper (from Institute of Cardiovascular Science, UCL) and surface plasmon resonance and computational docking Dr Jessica Holien, Dr Shiang Yong

Lim, Miss Naomi XY Ling and Dr Jonathan S Oakhill (from O'Brien Institute

Department, St Vincent's Institute of Medical Research).

I have dedicated this work to my mother, Soheila, who has been always there for me and helped me reach this level, and my father, Taher, who will be absent when I graduate but is always in my heart and will be always remembered. I owe my deepest gratitude to my brother and sister, Kaveh and Sara, and the rest of my family and friends in Iran and the United Kingdom, whose moral support were of high importance in the completion of this project.

7 List of Publications

Original Research and Review Articles

1. Beikoghli Kalkhoran S, Hall AR, White IJ, Cooper J, Fan Q, Ong S, Hernández‐Reséndiz S, Cabrera‐Fuentes H, Chinda K, Chakraborty B, Dorn GW, Yellon DM, Hausenloy DJ. Assessing the effects of mitofusin 2 deficiency in the adult heart using 3D electron tomography. Physiol. Rep. 2017 Sep;5(17):e13437. 2. Beikoghli Kalkhoran S, Munro P, Qiao F, Ong S-B, Hall AR, Cabrera-Fuentes H, Chakraborty B, Boisvert WA, Yellon DM, Hausenloy DJ. Unique morphological characteristics of mitochondrial subtypes in the heart: the effect of ischemia and ischaemic preconditioning. Discoveries 2017 Mar;5(1):e71. 3. Ong S-B, Beikoghli Kalkhoran S, Hernández-Reséndiz S, Samangouei P, Ong S-G, Hausenloy DJ. Mitochondrial-Shaping Proteins in Cardiac Health and Disease – the Long and the Short of It!. Cardiovasc. Drugs Ther. 2017 Feb;31(1):87–107. 4. Cabrera-Fuentes HA, Aragones J, Bernhagen J, Boening A, Boisvert WA, Bøtker HE, Bulluck H, Cook S, Di Lisa F, Engel FB, Engelmann B, Ferrazzi F, Ferdinandy P, Fong A, Fleming I, Gnaiger E, Hernández-Reséndiz S, Beikoghli Kalkhoran S, Kim MH, Lecour S, Liehn EA, Marber MS, Mayr M, Miura T, Ong S-B, Peter K, Sedding D, Singh MK, Suleiman MS, Schnittler HJ, Schulz R, Shim W, Tello D, Vogel C-W, Walker M, Li QOY, Yellon DM, Hausenloy DJ, Preissner KT. From basic mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and cardiac theranostics: meeting report from the third international symposium on “New frontiers in cardiovascular research”. Basic Res. Cardiol. 2016 Nov;111(6):69. 5. Hall AR, Burke N, Dongworth RK, Beikoghli Kalkhoran S, Dyson A, Vicencio JM, Dorn Ii GW, Yellon DM, Hausenloy DJ. Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction. Cell Death Dis. 2016;7(5):e2238. 6. Ong S-B, Samangouei P, Beikoghli Kalkhoran S, Hausenloy DJ. The mitochondrial permeability transition pore and its role in myocardial ischemia reperfusion injury. J. Mol. Cell. Cardiol. 2015;78:23–34. 7. Ong S-B, Hall AR, Dongworth RK, Beikoghli Kalkhoran S, Pyakurel A, Scorrano L, Hausenloy DJ. Akt protects the heart against ischaemia- reperfusion injury by modulating mitochondrial morphology. Thromb. Haemost. 2014 Sep;113(3):513–521. 8. Hussain A, Ghosh S, Beikoghli Kalkhoran S, Hausenloy DJ, Hanssen E, Vijay Rajagopal V. An Automated Workflow for Segmenting Single Adult Cardiac Cells from Large-Volume Serial Block-Face Scanning Electron Microscopy Data. J. of Struc. Biol. 2018; 202(3). 9. Beikoghli Kalkhoran S, Kriston-Visi J, Simoes Da Costa JR, Binte Katwadi K, Holien J, Ong SB, Y Lim S, Guet Khim MT, Arjun S, Chinda K, Samangouei P, Kwek X, Ketteler R, Yellon DM, Hausenloy DJ. A new pathway for Hydralazine induced Cardioprotection: The involvement of Mitochondrial Dynamics. (Unpublished).

8 Oral Presentations

1. Morphological Characteristics of Mitochondria in the Heart: The Effect of Ischaemic Preconditioning. Siavash Beikoghli Kalkhoran, Peter Munro, Fan Qiao, Sang-Bing Ong, Izzah Alfasihin, Andrew R.Hall, Bibhas Chakraborty, Derek M. Yellon, Derek J. Hausenloy. Singapore Cardiac Society, 2017. 2. Quantification of Mitochondrial Morphology and Its Interaction With Junctional Sarcoplasmic Reticulum in MFN2-KO Mice Using a 3 Dimensional Approach. Siavash Beikoghli Kalkhoran, Andrew R. Hall, Ian J. White, Jackie Cooper, Derek M. Yellon, Derek J. Hausenloy., ISHR, Bordeaux 2015.

Abstracts Presentations 1. S Beikoghli Kalkhoran, A R Hall, H Whittington, S M Davidson, D M Yellon, D J Hausenloy. Characterisation of Mitochondrial Morphology in the Adult Rodent Heart. Heart 2014;100:A2-A3. 2. S Kalkhoran, AR Hall, A Cole, White, DM Yellon, DJ Hausenloy. 3d Electron Microscopy Tomography to Assess Mitochondrial Morphology in the Adult Heart. Heart 2014;100:A10.

9 Table of Contents

Declaration ...... 2 Abstract ...... 4 Acknowledgements ...... 6 List of Publications ...... 8 Table of Contents ...... 10 Table of Figures ...... 14 List of Tables ...... 17 List of Abbreviations ...... 18 Chapter 1. Introduction ...... 20 Global Burden of Cardiovascular Diseases ...... 20 Mitochondrial Dynamics...... 21 1.2.1 Mitochondrial Fusion ...... 22 1.2.2 Mitochondrial Fission ...... 24 1.2.3 Regulators of Fission and Fusion ...... 26 Mitochondrial Fission and Fusion in Cellular Function ...... 28 1.3.1 Fission and Fusion Stability, Mitochondria Heterogeneity, and Maintenance of Mitochondrial Network Morphology ...... 28 1.3.2 Content Mixing and Mitochondrial Communication ...... 29 1.3.3 Mitochondrial DNA (mtDNA) Repair ...... 30 1.3.4 Metabolic Stress and Mitochondrial Dynamics...... 32 1.3.5 Fission and Fusion and Mitochondrial Culling ...... 32 1.3.6 The Relevance of Mitochondrial Dynamics in Cell Death ...... 34 1.3.7 Immunity ...... 35 1.3.8 Development and Mitochondrial Dynamics ...... 36 Mitochondrial Dynamics in the Adult Cardiomyocytes: Unique Subtypes of Cardiac Mitochondria ...... 37 1.4.1 Mitochondrial Subtypes: IMF and SSM Mitochondria ...... 39 1.4.2 Mitochondrial Subtypes: PN Mitochondria...... 42 Mitochondrial Dynamics in the Adult Cardiomyocytes: KO Mouse Models of Proteins That Participate in Mitochondrial Dynamics ...... 44 1.5.1 Animal Models of Proteins That Participate in Mitochondrial Fusion ...... 44 1.5.2 Animal Models of Proteins That Participate in Mitochondrial Fission ...... 47 Mitochondrial Dynamics in Health and Diseases ...... 49 1.6.1 Heart Failure (HF) ...... 49 1.6.2 Pulmonary Arterial Hypertension (PAH) ...... 50 1.6.3 Diabetic Mellitus (DM) ...... 51 1.6.4 Ischaemia Reperfusion Injury ...... 52 Methods to Assess Mitochondrial Dynamics...... 56 1.7.1 Electron Microscopy Assessment of Mitochondrial Morphology ...... 56

10 1.7.2 Light microscopy ...... 57 Chapter 2. Overall Aims and Objectives ...... 60 Main Hypothesis ...... 60 Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of Mitochondrial Morphology in Adult Cardiomyocytes ...... 60 2.1.1 Rationale: ...... 60 2.1.2 Aims and Objectives: ...... 61 Quantification of Mitochondrial Dynamics in 3D: Shape Descriptors of Mitochondrial Morphology in 3D ...... 61 2.2.1 Rationale: ...... 61 2.2.2 Aims and Objectives: ...... 61 High-throughput Screening of Small Molecule Modulators of Mitochondrial Dynamics ...... 62 2.3.1 Rationale: ...... 62 2.3.2 Aims and Objectives: ...... 62 Mitochondrial Dynamics and Hydralazine-Induced Cardioprotection ...... 63 2.4.1 Rationale: ...... 63 2.4.2 Aims and Objectives: ...... 63 Chapter 3. Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of Mitochondria in Adult Cardiomyocyte ...... 64 Introduction ...... 64 Methods ...... 65 3.2.1 Animals and Materials ...... 65 3.2.2 Ex-vivo Langendorff Characterisation Using Infarct Size ...... 66 3.2.3 Tissue Fixation Characterisation and Processing for TEM ...... 67 3.2.4 Ex-vivo Langendorff Model for TEM ...... 69 3.2.5 Image Acquisition and Analysis...... 70 3.2.6 Statistics ...... 73 Results...... 73 3.3.1 Characterisation of Langendorff Model Using the Infarct Size Assessment ...... 73 3.3.2 Examination of Heart Fixation Method ...... 74 3.3.3 Subpopulation Specific Differences in Mitochondrial Shape Parameters* ...... 75 3.3.4 Morphological Responses of IMF Mitochondria* ...... 79 3.3.5 Morphological Alterations of SSM Mitochondria after Ischaemia* ...... 82 3.3.6 Distinct Morphological Changes of PN Mitochondria after Ischaemia*...... 85 3.3.7 Morphological Shape Alterations of IMF Mitochondria in Mfn1/2 DKO ...... 88 Discussion ...... 91 Conclusion ...... 94 Chapter 4. Quantification of Mitochondrial Dynamics in 3D: Shape Descriptors of Mitochondrial Morphology in 3D ...... 95 Introduction ...... 95 Methods ...... 98 4.2.1 Materials/Mouse Strains ...... 98 4.2.2 Genotyping ...... 99

11 4.2.3 SBF- and FIB-SEM Imaging ...... 100 4.2.4 Sample Preparation and Imaging for STS-TEM ...... 100 4.2.5 3D Image Processing and Data Acquisition ...... 101 4.2.6 Statistics ...... 103 Results...... 104 4.3.1 Alteration of Mitochondrial 3D Shape Descriptors in Mfn2 KO Mitochondria ...... 104 4.3.2 Association of Mitochondrial 3D Shape Descriptors ...... 106 4.3.3 Anatomical Distribution of Mitochondria-jSR Network in Mfn2 KO Mice ...... 106 4.3.4 Evaluation of Mitochondrial Morphology and Network Structure Using SBF-SEM . 107 4.3.5 Evaluation of Mitochondrial Morphology and Network Structure Using FIB-SEM .. 111 Discussion ...... 115 Conclusion ...... 118 Chapter 5. High-throughput Screening of Small Molecule Modulators of Mitochondrial Dynamics ...... 119 Introduction ...... 119 Methods ...... 121 5.2.1 Cell Culture and Materials ...... 121 5.2.2 Image Acquisition for Algorithm Validation and Parameter Selection ...... 121 5.2.3 Cell Preparation for the Main Screen and Establishing the Dynamic Range ...... 122 5.2.4 Image Acquisition and Processing for the Main Screen ...... 123 5.2.5 Post-test Analysis and Drp1 GTPase Assay of Selected Compounds ...... 123 5.2.6 Statistics ...... 125 Results...... 125 5.3.1 Selection of Shape Descriptor of Interest ...... 125 5.3.2 Algorithm Output Comparison to Readout from Trained Human Eye ...... 128 5.3.3 Establishing the Dynamic Range for the Main Assay ...... 131 5.3.4 Screen Results of the mt-RFP Transfected HeLa Cells Treated with Compounds from Prestwick Library ...... 132 5.3.5 Post-test Analysis and Algorithm Output Verification ...... 134 5.3.6 Computational Docking for the GTPase Domain of Drp1 ...... 138 Discussion ...... 138 Conclusion ...... 142 Chapter 6. Mitochondrial Dynamics and Hydralazine Induced Cardioprotection ...... 143 Introduction ...... 143 Methods ...... 145 6.2.1 Animals and Cell Culture ...... 145 6.2.2 Dose-Response Determination of Hydralazine Concentration ...... 145

6.2.3 Characterisation of H2O2 Induced Fragmentation and Membrane Potential

Depolarisation and Hydralazine Impact on H2O2 Treated Cells ...... 146 6.2.4 Cardiac Cell Isolation ...... 146 6.2.5 Hypoxia Reoxygenation (HR) Model ...... 147 6.2.6 Evaluation of Mitochondrial Fusion Assay and Mitochondrial Morphology ...... 148 6.2.7 Cell Death ...... 149 6.2.8 Ex-Vivo Langendorff Infarct Model ...... 150

12 6.2.9 Hydralazine Effect on the GTPase Activity of His-Tagged Drp1 and Real-time Assessment of Hydralazine Binding to the His-tagged Drp1 Protein Using Surface Plasmon Resonance (SPR)...... 151 6.2.10 Statistics ...... 152 Results...... 152 6.3.1 Dose-Response Assessment of Mitochondrial Morphology in Cells Treated with Hydralazine ...... 152

6.3.2 Hydralazine Protects Against H2O2 induced Fragmentation ...... 154 6.3.3 Hydralazine Preserves Mitochondrial Dynamics in Cardiomyocytes Following HR Insult 159 6.3.4 Hydralazine Protects Against HR Induced Cell Death ...... 166 6.3.5 Ex-vivo Langendorff Hearts Undergoing IRI are Protected by the Action of Hydralazine ...... 167 6.3.6 Hydralazine Targeting of Drp1 and its Effect on Drp1 GTPase activity ...... 168 Discussion ...... 170 Conclusion ...... 173 Chapter 7. Summary and Future Work ...... 174 Chapter 8. References ...... 178 Chapter 9. Appendix ...... 212

13 Table of Figures

Chapter 1. Introduction

Figure 1-1 Schematic Presentation of Mitochondrial Fusion...... 23 Figure 1-2 Schematic Presentation of Mitochondrial Fission...... 25 Figure 1-3 Different Subpopulations of Cardiac Mitochondria...... 38

Chapter 3. Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of Mitochondria in Adult Cardiomyocyte

Figure 3-1 Delineation Example of Infarcted Area from Viable Myocardium in Control Group...... 67 Figure 3-2 Langendorff Ex-vivo protocol...... 69 Figure 3-3.Visual Description of Mitochondrial Shape Parameters...... 71 Figure 3-4. Morphometric Evaluation of Mitochondria with Disparate Shapes...... 72 Figure 3-5 Characterisation of Mouse Langendorff Model...... 74 Figure 3-6 Different Methods of Fixation and Their Impact on Cardiomyocytes. ... 75 Figure 3-7 Disparate Mitochondrial Subpopulations of Adult Mouse Cardiomyocytes...... 76 Figure 3-8 Morphometric Analysis of IMF, SSM and PN Mitochondria of Adult Cardiomyocytes...... 77 Figure 3-9 Distribution of Mitochondrial Morphometric Descriptors...... 78 Figure 3-10 Treatment Based Morphological Responses of IMF Mitochondria...... 79 Figure 3-11 Treatment Based Alterations of Shape Parameters in IMF Mitochondria...... 81 Figure 3-12 Treatment Based Distributions of IMF Mitochondrial Morphometric Descriptors...... 81 Figure 3-13 Treatment-Based Morphological Responses of SSM Mitochondria. .... 82 Figure 3-14 Treatment-Based Alterations of Shape Parameters in SSM Mitochondria...... 84 Figure 3-15 Treatment Based Distributions of SSM Mitochondrial Morphometric Descriptors...... 84 Figure 3-16 Treatment Based Morphological Responses of PN Mitochondria...... 85 Figure 3-17 Treatment Based Alterations of Shape Parameters in PN Mitochondria...... 87 Figure 3-18 Treatment Based Distributions of PN Mitochondrial Morphometric Descriptors...... 87 Figure 3-19 Alteration of Mitochondrial Morphology in MFN1/2 DKO Mice...... 88 Figure 3-20 Mitochondrial Shape Descriptors in WT and Mfn1/2 DKO Mice...... 89 Figure 3-21 Distributions of IMF Mitochondrial Morphometric Descriptors in WT and Mfn1/2 DKO Mice...... 90

14 Chapter 4. Quantification of Mitochondrial Dynamics in 3D: Shape Descriptors of Mitochondrial Morphology in 3D

Figure 4-1 2D and 3D Shape of an Adult Cardiac Mitochondrion...... 96 Figure 4-2 Example of Genotyped Samples...... 99 Figure 4-3 A Representative Reconstruction of Mitochondria, T-tubules and the j- SR...... 102 Figure 4-4 Mitochondria-jSR Distance Measurement...... 103 Figure 4-5 Influence of Mfn2 Ablation on Mitochondrial Shape Descriptors...... 104 Figure 4-6 Distributions of Mitochondrial 3D Shape Descriptors in WT and Mfn2 KO Mice...... 105 Figure 4-7 Influence of Mfn2 Ablation on Mitochondria-jSR Network Characteristics (from STS-TEM data)...... 107 Figure 4-8 Reconstruction of a Cardiac Myocytes...... 108 Figure 4-9 Mitochondria Network in Cardiac Myocytes...... 109 Figure 4-10 Autophagosome Formations along the Mitochondrial Network...... 110 Figure 4-11 3D Reconstruction of IMF from an adult cardiomyocyte using FIB- SEM...... 111 Figure 4-12 3D Reconstruction of SSM from an adult cardiomyocyte using FIB- SEM...... 112 Figure 4-13 FIB-SEM 3D Reconstruction of PN Mitochondria and Nucleus from an Adult Cardiomyocyte...... 113 Figure 4-14 3D Shape Descriptors of Different Subpopulation of Cardiac Mitochondria...... 114

Chapter 5. High-throughput Screening of Small Molecule Modulators of Mitochondrial Dynamics

Figure 5-1 Example of Docking Cluster for Zimelidine and Phenformin Hydrochloride...... 124 Figure 5-2 Representative Confocal Images of Mitochondria in Hela Cells for Each Morphological Category...... 126 Figure 5-3 Comparison of Frequency Distribution of Shape Descriptors of Different Mitochondrial Population...... 127 Figure 5-4 Comparison of Roundness Frequency Distribution in Cells with Different Mitochondrial Population*...... 130 Figure 5-5 Comparison of Roundness Frequency Distribution in Cells After Individual Treatments...... 131 Figure 5-6 Summary of Z'-Factors for Individual Replicates...... 132 Figure 5-7 Images from Two Ends of Z-score List...... 133

15 Chapter 6. Mitochondrial Dynamics and Hydralazine Induced Cardioprotection

Figure 6-1 HR Protocol in Isolated Cardiomyocytes...... 147 Figure 6-2 Tracking Mitochondrial Fusion Dynamics in Adult Cardiomyocytes. . 149 Figure 6-3 Langendorff Protocol for the Assessment of Infarct Size in Hearts Treated with Hydralazine or Veh. CT...... 150 Figure 6-4 Response of Mitochondrial Morphology to Different Dosages of Hydralazine...... 153 Figure 6-5 Effect of Different H2O2 Incubation Time on Mitochondrial Morphology and Membrane Potential...... 155 Figure 6-6 Effect of Different H2O2 Incubation Time on Mitochondrial Morphology...... 156 Figure 6-7 Hydralazine Induced Preservation of Mitochondrial Shape and Membrane Potential after H2O2 Insult...... 157 Figure 6-8 Preservation of Mitochondrial Morphology by Hydralazine in H2O2 Treated Cells ...... 158 Figure 6-9 Hydralazine Induced Preservation of Mitochondrial Morphology in HR Treated Cardiomyocytes ...... 159 Figure 6-10 Fusion Events after Photo-activation in Normoxic Time CT. Group. 161 Figure 6-11 Mitochondrial Fusion Characteristics in Untreated (Normoxic Time CT.) Cardiomyocytes...... 162 Figure 6-12 Fusion Events in Veh. CT. Group after HR...... 163 Figure 6-13 Fusion Events in Hydralazine Group after HR...... 164 Figure 6-14 Mitochondrial Fusion Events for Individual Subpopulations after Each Treatment...... 165 Figure 6-15 Cell Death Assessment for Different Treatment Groups Using PI Staining...... 166 Figure 6-16 Infarct Size in Hearts Treated with Either Veh. CT or Hydralazine. .. 167 Figure 6-17 Example of Flow Cell No.4 SPR Binding Plot...... 168 Figure 6-18 GTPase Activity of Drp1 in the Presence of Different Dosages of Hydralazine...... 169

16 List of Tables

Chapter 3. Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of Mitochondria in Adult Cardiomyocyte Table 3-1 Quantification of Mitochondrial Shape Parameters in Different Subpopulations of Cardiac Mitochondria Using Predicted Mean Values ± S.E. (Modified from (277))...... 76 Table 3-2 Treatment Based Evaluation of IMF Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277))...... 80 Table 3-3 Treatment Based Evaluation of SSM Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277))...... 83 Table 3-4 Treatment Based Evaluation of PN Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277))...... 86

Chapter 5. High-throughput Screening of Small Molecule Modulators of Mitochondrial Dynamics

Table 5-1 Comparison between the output of mitochondrial morphology readouts from the algorithm and human eye...... 129 Table 5-2 Nominated 10 Drugs from Top 20 Drugs Causing Mitochondrial Elongation/Inhibiting Fragmentation ...... 134 Table 5-3 Nominated 10 Drugs from Top 20 Drugs Causing Mitochondrial Fragmentation/Inhibiting Elongation...... 135 Table 5-4 Re-Scoring the Images Using Human Eye for Drugs Causing Mitochondrial Elongation/Inhibiting Fragmentation ...... 136 Table 5-5 Re-Scoring the Images Using Human Eye for the Drugs Causing Fragmentation/Inhibiting Elongation...... 137 Table 5-6 The Docking Scores of Top 20 Drugs Causing Elongation/Inhibiting Fragmentation ...... 139

Chapter 6. Mitochondrial Dynamics and Hydralazine Induced Cardioprotection

Table 6-1 Statistical Parameters of the Effect of Different Dosages of Hydralazine on Mitochondrial Roundness ...... 154 Table 6-2 Summary of Hydralazine Binding Kinetics to the Drp1 Protein...... 169

Chapter 9. Appendix

Table 9-1 The HTS Results. Drugs Corresponding to the Plate and Position Number Can be Found in Table 9-10...... 212 Table 9-2 List of Drugs in Prestwick Chemical Library. For the Z-score of Individual drugs Refer to Table 9-1...... 222

17 List of Abbreviations

Cardiovascular diseases (CVDs)

Charcot–Marie–Tooth disease type 2A (CMT2A)

Connexin-43 (Cx43)

Corrected total cell fluorescence (CTCF)

Coronary heart disease (CHD)

Diabetes mellitus (DM)

Dynamin-related protein 1 (Drp1)

Embryonic stem cells (ESCs)

Empirical cumulative distribution functions (ECDF)

Endoplasmic reticulum (ER)

Focused ion beam (FIB)

Heart failure (HF)

Human epithelial carcinoma (Hela)

Hypoxia Reoxygenation (HR)

Inner mitochondrial membrane (IMM)

Intermyofibrillar (IMF)

Ischaemia/reperfusion injury (IRI)

Ischaemic preconditioning (IPC)

Knock out (KO)

Left ventricular hypertrophy (LVH)

Mfn1 and Mfn2 Double KO (Mfn1/2 DKO)

Maximal deviation value (d-value)

Mitochondria division inhibitor-1 (Mdivi-1)

Mitochondrial DNA (mtDNA)

Mitochondrial fission factor (Mff)

Mitofusin 1 (Mfn1)

18 Mitofusin 2 (Mfn2)

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)

Outer mitochondrial membrane (OMM)

Perinuclear (PN)

Presenilin-associated rhomboid-like (PARL)

Protein Kinase A (PKA)

Pulmonary arterial hypertension (PAH)

Reactive oxygen species (ROS)

Repeatability Standard Deviation (RSD)

Resonance unit (RU)

Sarcoplasmic reticulum (SR)

Serial block face scanning electron microscopy (SBF-SEM)

Serial thin section (STS)

Stabilisation (ST)

Stress-induced mitochondrial hyper fusion (SIMH)

Subsarcolemmal (SSM)

Super-resolution microscopy (SRM)

Surface Plasmon Resonance (SPR)

Transmission electron microscopy (TEM)

Triphenyltetrazolium chloride (TTC)

Vehicle control (Veh. CT.)

Wild-type (WT)

19 Chapter 1. Introduction

Global Burden of Cardiovascular Diseases

The global burden of cardiovascular diseases (CVD) remains alarming worldwide (1). In 2016, CVD accounted for more than one-third of all mortality in

Europe and USA with numerous factors such as ethnicity, ageing population, diet, lifestyle, and social class contributing to its high prevalence (2; 3). Among all major causes of CVD, coronary heart disease (CHD), remains one of the leading causes of mortality and morbidity (1; 4). In the UK, it was estimated that 19% and 10% of total mortality in men and women in 2012, respectively, were due to CHD (5). The same study found that the total population suffering from CHD was estimated to be around

2.3 million with males being three times more prone to myocardial infarction than females (5). Interestingly, a recent publication by the same group indicated that the occurrence of total death related to CVDs and CHD - declined in the UK until 2014.

However, they showed a surge in the number of treatments and the incidence of hospital admissions, especially for heart failure (HF) (6).

Identification of new treatments for protecting the myocardium is, therefore, necessary to improve clinical outcomes in patients with CHD. In this regard, emerging data have implicated mitochondria as important targets for cardioprotection.

Mitochondrial function during acute myocardial ischaemia/reperfusion injury (IRI) is a critical determinant of cardiomyocyte death. Recent experimental data have implicated mitochondrial dynamics, a phenomenon which critically impacts on mitochondrial function, as a target for cardioprotection (7–9). Emerging studies suggest that it may also be relevant to other CVDs such as pulmonary hypertension

20 (10) HF (11; 12), diabetic cardiomyopathy (13), endothelial dysfunction (14) and left ventricular hypertrophy (LVH) (15).

In order to maintain a healthy mitochondrial network, mitochondria change their morphology by undergoing either fusion (forming elongated mitochondria) or fission (forming fragmented mitochondria), under the regulation of the fusion proteins

(Mfn1, Mfn2, Opa1) and fission proteins (Drp1, hFis1, Mff, MiD49/51), respectively

(16). Until recently, the investigation of mitochondrial dynamics had been largely confined to non-cardiac cells, in which mitochondria are free to move and change their shape. However, the relevance of mitochondrial dynamics in the three mitochondrial subpopulations (interfibrillar, subsarcolemmal, perinuclear) of the adult heart, in which mitochondria appear to be static, have been elusive (17). In this chapter, we primarily focus on the nature of mitochondrial dynamics and its relevance to the adult heart. The main research questions and aims of this thesis will be introduced in the concluding section of this chapter.

Mitochondrial Dynamics

Mitochondria are one of the key components of eukaryotic life. Their roles in metabolism and energy generation are more pronounced in cells such as cardiomyocytes, which require a constant supply of energy. These organelles have unique dynamics and change shape by undergoing fission and fusion upon alterations in metabolic requirements (17; 18). It should be noted, that the term “dynamics” is not limited to the fission and fusion processes and is comprehensively used to refer to mitochondrial morphological changes, content mixing and communication, and the movement of mitochondria inside cells. The fission and fusion processes that govern mitochondrial dynamics mediate vital tasks such as mitochondria distribution within

21 cells and maintenance of mitochondrial network morphology (19; 20); content mixing and communication (21; 22); mitochondrial DNA (mtDNA) repair (23); adaptation to metabolic demands (18; 24; 25); elimination of dysfunction mitochondria by mitophagy (26; 27); and cell death (28). Here we review some of the fundamental principles of these processes.

1.2.1 Mitochondrial Fusion

Expansion of the mitochondrial network within mammalian cells occurs via the process of fusion (Figure 1), which is essential for the health and distribution of mitochondria inside the cell (29). Fusion begins with the actions of mitofusins 1 and 2

(Mfn1 and Mfn2, respectively). These two outer mitochondrial membrane (OMM) proteins form heterotypic Mfn1-Mfn2 trans or homotypic Mfn1 or Mfn2 cis complexes to induce mitochondrial OMM fusion (30; 31). Mitofusins facilitate OMM fusion by GTP hydrolysis. Although Mfn1-Mfn1 oligomer complexes are more efficient at hydrolysing GTP, the trans Mfn1-Mfn2 complexes have greater fusion activity (31; 32). OMM fusion ultimately occurs when MITOPLD finalises membrane aggregation (33).

The fusion of OMM between two neighbouring mitochondria is followed by the inner mitochondrial membrane (IMM) fusion, which begins with the action of the dynamin-related GTPase, optic atrophy 1 (Opa1) (34). After the import into mitochondria, the N-terminal sequence of Opa1 protein becomes cleaved to produce long Opa1 isoforms in the IMM (35). The long isoforms undergo proteolytic cleavage in the mitochondrial matrix by Oma1 at S1 site, and Yme1L at S2 site to produce the short Opa1 (34; 36). Concurrently, homotypic L-Opa1/L-Opa1 or heterotypic L-

Opa/S-Opa dimers mediate IMM fusion (37). This step is accompanied by an increase

22 in the level of oxidative phosphorylation, which contributes to the coupling of OMM and IMM and completes the fusion of two mitochondria (38). Unlike mitochondrial fission which is dependent on actin cytoskeleton(39), mitochondrial fusion occurs independently from cytoskeleton; however, destabilising the integrity of actin cytoskeleton can hamper the fusion rate (40).

Figure 1-1 Schematic Presentation of Mitochondrial Fusion. This diagram illustrates various phases of the mitochondrial fusion process. Fusion begins with the formation of cis- and trans-mitofusin complexes on the surface of OMM (30; 31). Following this event, MITOPLD brings the membranes closer by utilising cardiolipin and ultimately completes the OMM fusion (33). At the same time, long and short forms of Opa1 create homo- and heterotypic complexes to allow IMM fusion (34; 36).

23 1.2.2 Mitochondrial Fission

Mitochondrial fission (Figure 2) is equally important as mitochondrial fusion and is involved in vital cellular functions including the division of mitochondria during cell proliferation and the selective removal of damaged mitochondria by mitophagy

(41–43). The GTPase, Dynamin-related protein 1 (Drp1), is the key modulator of mitochondrial fission. Once inactive cytosolic Drp1 becomes active, through the dephosphorylation of its Ser-637 domain, it translocates to the OMM (44). The N-

Terminus of human fission factor 1 (hFis1) and/or mitochondrial fission factor (Mff) then bind to Drp1 on the surface of OMM (45; 46). Mitochondrial dynamics 49 and

51 kDa (MiD49 and MiD 51, respectively) also engage in the recruitment of Drp1 to the surface of OMM (47) and act as adapters for Mff-Drp1 association (48). The contribution of MiD51 to Mff-Drp1 linkage is higher than MiD49 (49). The MiD49/51 proteins can also act independently of Mff and hFis1 to directly recruit Drp1, thereby acting as OMM receptors for Drp1. In this regard, MiD 49 binding efficiency to Drp1 is higher than MiD51 (50).

Upon binding to the OMM, Drp1 forms a helical structure around the mitochondria

(51) at the endoplasmic reticulum (ER)-mitochondria contact sites (52). More specifically, inverted formin 2 and F-actin facilitate Drp1 helix formation around the fission site (39; 53). This process then follows the recruitment of myosin IIA and IIB heavy chains at the Drp1 puncta (54). Myosin IIA specifically facilitates a Ca2+ increase at the mitochondrial associated ER-membrane (55). Co-ordinately, Yme1L increases the generation of S-Opa1 which dissociates the MIC60/Mitofilin, in a Ca2+- dependent manner. This process together with the action of myosin proteins allows the constriction of IMM (55–57). At this stage, Drp1 hydrolyses mitochondrial cardiolipin, which is located in the IMM, to induce the formation of constriction

24 tubules around the mitochondria (44). This process involves the detection of cardiolipin by the Drp1 variable domain which allows the assembly of the Drp1 middle and GTPase effector domain. Dimerisation of the Drp1 GTPase-domain, which governs Drp1 GTPase activity, ultimately finishes the process of fission (58).

Figure 1-2 Schematic Presentation of Mitochondrial Fission. This diagram illustrates the distinct stages of the mitochondrial fission process. Fission begins with the recruitment of the active form of Drp1 to the surface of OMM via Mff/MiD51, hFis1 or MiD49/51 dependent manner (47; 49). Subsequently, the MIC60/mitofilin complex is dissociated via S-Opa (56) thereby preparing the IMM for fission. Drp1 then hydrolyses cardiolipin (44) and with the functional contribution of F actin (53) and formin 2 (54) forms a helix around the mitochondrion after which complete fission ensues.

25 1.2.3 Regulators of Fission and Fusion

Maintenance of cellular homeostasis requires the tight regulation of mitochondrial fission and fusion activity, and this is influenced by the post-translational modification of mitochondrial fission and fusion proteins (59).

1.2.3.1 Regulation of Mitochondrial Fusion Proteins

Cellular levels of the mitochondrial fusion proteins are regulated by

Peroxisome proliferator-activated receptor gamma coactivators 1-alpha and 1-beta

(PGC-1α and PGC1-β, respectively) (60; 61). PGC1-α mutant mice have decreased

Opa1 and Mfn1 expression (61), whereas deficiency in PGC1-β reduces the expression of Opa1 and both Mfn1 and Mfn2 proteins (60).

Following transcription, the modification of mitochondrial fusion proteins becomes more complex. Human epithelial carcinoma (HeLa) cells lacking MIB protein have been shown to exhibit extensive mitochondrial fusion. This mechanism is hampered in the absence of MIB implying that the expression of Mfn1 under basal conditions is governed by the MIB protein (62). In an elegant study, Karbowski et al. (2006) showed that mitochondrial fragmentation in cells lacking the apoptotic factors, Bak and Bax, can be rescued by the re-expression of Bax and Mfn2, revealing the regulatory action of Bax on Mfn2. The same study also documented that the Bax protein was responsible for the localisation and foci formation of Mfn2 (63). In comparison to mitofusins, cellular levels of Opa1 are regulated by Presenilin- associated rhomboid-like (PARL). Mouse embryonic fibroblasts devoid of PARL have a reduced intermembrane soluble form of Opa1 and are more susceptible to cell death when challenged with apoptotic stimuli (64).

26 Mitochondrial fusion proteins are also modified during stress conditions (65).

For example, during the process of mitophagy or hypoxic stress, mitofusin proteins are ubiquitinated and marked for degradation by Parkin/PINK1 and MARCH5/MITOl proteins, respectively (66–68). In addition, OMA1, which is essential for maintenance of Opa1 under basal levels, becomes upregulated after stress to induce cleavage of L-

Opa1 and permit the release of cytochrome C (69). In contrast, stress-induced activation of Sirt3 induces the hyper deacetylation of Opa1 thereby preserving its fusion activity and preventing cell death (70).

1.2.3.2 Regulation of Mitochondrial Fission Proteins

In parallel to the mitochondrial fusion proteins, PGC-1α also controls the transcription of fission proteins including Drp1 and hFis1 (61), whereas miR-761 governs the gene expression of Mff (71). There is limited data on the transcriptional regulation of MiD49/51.

Under basal conditions, Drp1 remains phosphorylated at 637 domain and dephosphorylated at 616 domain - these sites then change their status prior to the initiation of mitochondrial fission (72–74). These domains are highly regulated by the action of proteins such as calcineurin, Rho-associated coiled coil-containing protein kinase-1, Pim-1, and protein kinase A (PKA) (73–76). Proteolytic modification of

Drp1 is not limited to phosphorylation. Drp1 ubiquitination by March V is vital for mitochondrial fragmentation and cell growth and mutant March V HeLa cells show extensive mitochondria fusion and senescence (77). March V also ubiquitinates

MiD49 to facilitate the induction of cell death (78; 79). The fission function of Drp1 is additionally maintained by the SUMOylation via Ubc9 as well as MAPL or diminished by deSUMOylation via SNEP5 proteins (80–82). Specifically, the loss of

MAPL destabilises mitochondria ER contact sites thereby preventing the formation of

27 the Drp1 helix that is required for fission induced apoptosis (80). Furthermore, Drp1 activity increases while undergoing O-GlcNAcylation in diabetic mice or S- nitrosylation during neurodegeneration (83; 84) thereby revealing the post-translation complexity of Drp1 protein. Whether therapeutic approaches should solely target

Drp1, Drp1 regulators, or both remains an open question. It is also noteworthy to mention that the function of Mff is also affected by proteolytic modification. Protein members of autophagy such as Parkin and AMPK ubiquitinate or phosphorylate Mff, respectively, in order to trigger fission linked mitophagy (85; 86). These discoveries highlight the close link between mitochondrial fission and cell death and reveal the interdependence of these processes to the diverse regulation of Drp1. They also pinpoint the opposing impact of post-translational modification on fusion versus fission proteins.

Mitochondrial Fission and Fusion in Cellular Function

1.3.1 Fission and Fusion Stability, Mitochondria Heterogeneity, and

Maintenance of Mitochondrial Network Morphology

The balance between fission and fusion influences the mitochondrial network morphology in various cell types (87) and allows the cellular needs to be met (19; 88).

The extent of occurrence of fission and fusion events varies in different cells and is dependent on cellular metabolism (89). A study in mouse embryonic fibroblasts

(MEF) and HeLa cells showed that the process of fission is often followed by fusion or fission in a paired or unpaired manner, respectively. These selective dynamics are highly dependent on the function of Mfn1. The occurrence of these events correlate with mitochondrial respiration and movement (88) and allow the elimination of dysfunctional mitochondria (27) thereby indicating the importance of stability between fission and fusion events.

28 In the presence of balanced fusion, mitochondria with dominant elongated morphology are present in the cytosol and are more populated in the perinuclear (PN) region (30; 90; 91). Tubular mitochondria are often present in transformed and primary cells lines, but exceptionally some cells such as hepatocytes exhibit shorter less elongated network morphology (87). In contrast to a balanced fusion status, higher fission events mediate a decrease in the density of mitochondria around the PN region and shift the distribution of mitochondria towards cellular regions that were devoid of mitochondria under balanced fusion (91). Modulation of mitochondrial fission or fusion by knockdown of Drp1 or Opa1 mutations promote the accumulation of mitochondria in the PN region of primary neuronal cells. The imbalance in mitochondrial distribution then leads to disturbed neuronal function (19). These facts demonstrate that mitochondrial dynamics vary across different cells and shows the interconnectivity between cellular function and mitochondrial network distribution.

1.3.2 Content Mixing and Mitochondrial Communication

Mitochondria have adopted complex communication routes to respond to alterations in cellular environment (22). Using mitochondrial targeted mtGFP or mtRFP probes, Hajnoczky et al. (2009) demonstrated complete and transient fusion events in H9C2 cells. They showed that the mitochondrial fusion events depend on mitochondrial cytoskeleton as well as mitochondrial membrane potential. Only 28% of the identifiable fusion events were of complete fusion. The remaining events were of transient nature (termed as “kiss and run”) where partial and reversible fusion is preferred to complete fusion (22; 92). Transient fusion is not unique to mitochondria- mitochondria communication and is also observed during the processes of exo- and endocytosis where the short intra-vesicle molecular transfer takes place (93). An

29 interesting example of this process is seen in mitochondria from erythrocytes which use ‘kiss and run’ to transfer iron from endosomes (94).

The essence of selection between complete and transient fusion is highly affected by physiological and pathophysiological factors. For instance, enlarged mitochondria that are formed by the inhibition of mitophagy in rat myoblasts do not undergo complete fusion (95). Comparatively, mitochondria rely on transient fusion to deliver tBid-Bax- induced apoptotic signals throughout the cell (96). Furthermore, these two main types of fusion occur between adjacent mitochondria whilst fusion in cardiac mitochondria is not always limited to the neighbouring mitochondria. In fact, cardiac mitochondria have developed a more complex communication method known as “nanotunneling”, where they content-exchange via protruding tubules with mitochondria that are located outside their proximity (97). This behaviour is regulated by the function of Ryanodine receptors and depends on Ca2+ availability (97). The protein(s) which facilitate the protrusion of tubules from cardiac mitochondria is unknown. KIF5B is a protein which mediates mitochondrial network formation in mammalian cells (98). KIF5B also regulates mitochondrial localisation. KIF5B KO mice have muscular dystrophy and their myoblasts have high aggregation and accumulation of mitochondria around their cell nucleus (99). Whether KIF5B participate in mitochondrial dynamics in adult cardiomyocytes is yet to be assessed. Hence, further studies are necessary to study mitochondrial content exchange and identify their key mediators.

1.3.3 Mitochondrial DNA (mtDNA) Repair

The integrity of mtDNA is vital for its function (23; 100; 101). mtDNA is more prone to mutations that are induced by environmental factors in comparison to cellular nuclear DNA (102). One of the main roles of mitochondrial dynamics is to preserve

30 mtDNA function and compensate for the genomic mutations (23; 103). There is evidence that complete fusion allows the functional complementation of mtDNA (104;

105), but whether transient fusion also impacts on the mtDNA integrity has not been shown. In line with these facts, the ablation of both mitochondrial fission and fusion proteins, as well as autophagic markers in C. elegans, inhibit the removal of mutations induced by ultraviolet C radiation. Interestingly, the ultraviolet C radiation-induced mutations do not alter mitochondrial morphology (102). In parallel, mice lacking Mfn1 and Mfn2 proteins have a 14-fold reduction in mtDNA copies and exhibit severe post- natal developmental deformities (23). Likewise, abrogated Opa1 expression in patients suffering from mitochondrial respiratory chain deficit has been linked with mtDNA defects (106). In comparison to mitochondrial fusion proteins, ablation of hFis1 or Drp1 in human ρ0 rhabdomyosarcoma cells also causes a loss of mtDNA copy number and results in the increase in mtDNA point mutations (107). Hence, fission, as well as fusion, are required to preserve a healthy mtDNA by eliminating potential mutations.

Whether the rate of mitochondrial fission and fusion also play a role in mtDNA integrity is controversial (108). Using mathematical modelling Figge et al. (2012) showed that the deceleration in the rate of mitochondrial fission and fusion can preserve mtDNA in the ageing process (109). In contrast, a separate mathematical model focusing on the role of mitochondrial dynamics in the maintenance of mtDNA showed that the slower rate of fission and fusion increases the propensity for the accumulation of mtDNA mutations (108). The fundamentals of the rate of fission and fusion in pathological conditions are poorly understood and it would be intriguing to know whether their alteration can offer any therapeutic benefits.

31 1.3.4 Metabolic Stress and Mitochondrial Dynamics

Cell function under pathophysiological stress relies on the performance of mitochondrial fission and fusion (110). One of the most common mitochondrial stress responses is the “stress-induced mitochondrial hyper fusion (SIMH)” which acts as an inhibitory mechanism against autophagy and downstream apoptosis (24; 111). Various cell lines including MEFs, HeLa and hepatocytes mitochondria form an extensive network of tubules under the influence of different stress inducers such as UV irradiation, RNA transcription inhibition, amino acid starvation and serum deprivation.

The inhibitory action of Stomatin-like protein 2 (SLP-2) on proteolytic cleavage of L-

Opa1 is the key factor for the occurrence of SIMH (111). Besides, upregulated phosphorylation of Drp1 at the Ser-637 domain by PKA and the reduction of its phosphorylated form at the Ser-616 site are also involved in the induction of SIMH

(18; 24).

On the contrary, mitochondria also fragment during stress conditions such as oxidative stress which is seen mostly in neurodegenerative diseases (112). The induction of oxidative stress often mediates a fall in mitochondrial membrane potential which subsequently activates Drp1 (113). Correspondingly, other pathophysiological stressors such as glutamate excitotoxicity induce the upregulation of glutamate receptors which facilitate a surge in oxidative stress and downstream mitochondrial fragmentation (114).

1.3.5 Fission and Fusion and Mitochondrial Culling

The optimal cellular function is achieved by the elimination and recycling of dysfunctional mitochondria in a process known as mitophagy (115). Under stress conditions, damaged mitochondria that have lost their membrane potential are

32 selectively removed (116; 117). This process begins with the translocation of cytosolic protein PTEN-induced putative kinase 1 (PINK1) to the surface of the depolarised mitochondria (86). At the OMM, PINK1 has been shown to phosphorylate Mfn2 which then stimulates the cytosolic to mitochondrial translocation of the main mitophagic protein, Parkin. Subsequently, Parkin ubiquitinates MFN2 and allows its degradation

(119). During mitophagy, the Mfn1 protein is also ubiquitinated by Parkin translocation (116). In addition, dermal fibroblasts lacking functional Opa1 obtained from patients suffering from dominantly inherited optic atrophy exhibit higher accumulation of mitophagosomes, (120) suggesting the initiation of mitophagy requires the downregulation of all major fusion components.

On the other hand, the activation of fission is a necessary step for the execution of mitophagy. Complete or partial ablation of Drp1 induces the formation of interconnected mitochondrial phenotype which attenuates the process of mitophagy in primary cell lines (121; 122). In contrast, complete ablation of Drp1 in the heart promotes an overwhelming production of Parkin which then triggers excessive mitophagy which eventually leads to cardiomyopathy (123). Moreover, the overexpression of hFis1 induces mitochondrial fragmentation and increases the population of autophagic markers (124) further emphasising that the status of mitochondrial morphology governs the process of mitophagy.

The process of fusion is also important for the initiation of mitophagy under basal non-stress related conditions. Often, mitochondria that are produced after the mitochondrial fission have unbalanced mitochondrial membrane potential and hence those mitochondria with higher membrane potential are selected for fusion; whereas, those mitochondria with lower membrane potential undergo mitophagic removal (27).

Correspondingly, mitochondria that are capable of fusion have been shown to be able

33 to avoid the mitophagic culling (18). Conversely, the formation of hyperfused mitochondrial network initially protects against stress-induced mitochondrial damage

(111), but as damage ensues, the inhibition of mitochondrial fusion machinery is initiated to allow the induction of mitophagy (116; 125). Taken together, these results indicate the onset of mitophagy is more dependent on mitochondrial fusion rather than mitochondrial fission under basal conditions and this process is reversed during stress.

1.3.6 The Relevance of Mitochondrial Dynamics in Cell Death

The dynamics of mitochondria are closely linked with the core aspects of apoptosis (126). In mammalian cells, pro-apoptotic signals initially enable the recruitment of Drp1 to the surface of mitochondria (127; 128). Upon stimulation with apoptotic mediators, COS-7 cells, transfected with mitochondrial targeted GFP, exhibit fragmented and rounded mitochondria due to translocation of cytosolic Drp1 to mitochondria (129). The recruitment of Drp1 to the OMM is achieved by the mitochondrial fission machinery hFis1, Mff and MiD49/51 (127; 130; 131). The Drp1 recruitment process follows the activation of apoptotic factors including Bak and Bax proteins that translocate to mitochondrial surface to enable OMM permeabilisation, a process which increases the fission-inducing activity of Drp1 (129; 132). The increase in Drp1 levels after Bak and Bax recruitment is also partially due to the sumoylation of small ubiquitin-like modifier-1 (SUMO-1) on the surface of Drp1 (133). After Drp1 translocation, remodelling of mitochondria cristae begins with Romo1 and Opa1 downregulation in turn results in the release of cytochrome C and cell death (127; 134;

135). The function of Opa1 in cristae remodelling has been suggested to be independent of its fusion activities (136). Apoptosis can also occur in a Drp1- independent manner via the action of hFis1. This form of apoptosis relies on both Bak and Bax and MEF cells devoid of these proteins have Ca2+-related ER deficiency that

34 hinders apoptosis (137). Mitofusins also participate in the induction of apoptosis (138).

Endogenous Bak is usually associated with Mfn2 and after the release of an apoptotic signal, it dissociates from Mfn2 (138). The dissociated Bak then binds to the phosphorylated Mfn1 and inhibits its fusion activity (139). Concomitantly, Mfn1 also becomes downregulated by the action of apoptotic MicroRNA-140 thereby further compromising the process of fusion (140). In comparison to Mfn1, the exact behaviour of the Mfn2 protein in apoptosis is controversial. After receiving the apoptotic signal,

Mfn2 becomes phosphorylated at the Ser-267 site. Upon phosphorylation, Mfn2 loses its fusion activity and becomes susceptible to ubiquitination and proteolytic cleavage

(141). In favour of this finding, Slack et al. (2007) showed that the over-activation of

Mfn2 can protect against oxidative and DNA damage-induced apoptosis (142). In contrary, the overexpression of Mfn2 in vascular smooth muscle cells was shown to induce apoptosis, independent of its pro-fusion role (143) thereby alluding to the pleiotropic and cell-specific function of the Mfn2 protein. Thus, the coordinated regulation of mitochondrial shaping proteins is fundamental to the execution of apoptosis.

1.3.7 Immunity

Mitochondrial dynamics has been shown to play a role in immunity (144).

Mitochondrial fission via Drp1 has been shown to mediate the upregulation of inflammatory transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), which is known to attract immune cells such as natural killer T cells and effector T cells (145–147). Mfn1 and Mfn2 also participate in immune cell migration by activating RIG-I-like receptor which promotes the activation of pro-inflammatory Interferon β (148). However, a previous report by the same group had indicated that the loss of Mfn2 triggers pro-inflammatory Interferon β

35 activation (149). Upon activation, circulating T cell effector lymphocytes, which possess very low energy demand, maintain a Drp1-dependant fragmented mitochondrial network. Conversely, cross-differentiation of effector T cells into memory T cells requires a fused network of mitochondria due to higher oxidative phosphorylation status. T cells accomplish this task by the upregulating the Opa1 expression (146). Although mitochondrial fission and the main action of Drp1 seems indispensable for the function and activation of immune cells, the exact role of mitochondrial fusion proteins in these processes is poorly understood and remains an open avenue for future research.

1.3.8 Development and Mitochondrial Dynamics

Mitochondrial dynamics are also vital for development and growth.

Dysfunction of fusion proteins gives rise to several metabolic defects that are detrimental to organ development. The most well-known example is the Charcot–

Marie–Tooth disease type 2A (CMT2A) which is a form of peripheral neuropathy that occurs due to a loss-of-function mutation in Mfn2 (150). CMT2A associated mutation of Mfn2 disturbs the transport of axonal mitochondria and hence leads to neurodegeneration (151). Relatively, loss of Mfn2 in pluripotent stem cells inhibits neuronal maturation and metabolic development of cardiac cells (152; 153).

Homozygous knock out (KO) of either Mfn1 or Mfn2 results in embryonic death which is mainly due to a placental defect (30), suggesting that mitofusins are required for embryonic development.

Optic atrophy is another example of a defect caused by a loss-of-function mutation in Opa1 gene. Homozygous KO forms of Opa1 are embryonically lethal, and mice carrying heterozygous forms have impaired mitochondrial fusion and respiratory

36 chain defects (154). MITOPLD has been also shown to participate in cell growth and germline mutation of this protein promote meiotic arrest thereby causing growth defects (155).

The impact of mitochondrial fission proteins on cellular development resemble the effects exerted by the absence of fusion proteins. Loss of Drp1 is embryonically lethal and prevents the normal brain and heart development (156; 157).

On the other hand, overexpression of Drp1 alters mitochondrial dynamics and mtDNA leading to the inhibition of muscle growth (158). In contrast, upregulation of Drp1 has been shown to be correlated with the formation of malignant oncocytic thyroid tumours in human patients (159), thereby suggesting that the function of Drp1 in cell development differs based on the nature of pathophysiological conditions.

Similar to the function of Drp1, downregulation of mitochondrial OMM localised protein, hFis1, results in the formation of irregular cell morphology and abnormal cellular structure (160). This ultimately leads to the induction of senescence and cell cycle arrest (161). Moreover, diminished Mff function has been shown to correlate with neuropathy in patients suffering from Leigh-like basal ganglia disease

(162). These findings reflect that any alteration in fission and fusion process can drastically alter the normal development.

Mitochondrial Dynamics in the Adult Cardiomyocytes: Unique Subtypes of

Cardiac Mitochondria

The evolving topic of mitochondrial dynamics has become a major focus in the field of cardiovascular research (163; 164). However, the study of mitochondrial dynamics in the adult heart has been challenging and up to recently, the focus was mainly on non-cardiac and cardiac cell lines (163). Mitochondria from adult

37 cardiomyocytes are characteristically arranged into 3 subpopulations: subsarcolemmal

(SSM) mitochondria which reside below the plasma membrane of the cell; intermyofibrillar (IMF) mitochondria that are positioned between the myofibrils, and

PN mitochondria that are localised adjacent to the nucleus as shown in Figure 1.3 A and B.

Figure 1-3 Different Subpopulations of Cardiac Mitochondria. (A) original and (B) segmented electron micrographs of the three subpopulations of cardiac mitochondria including intermyofibrillar (IMF), subsarcolemmal (SSM) and perinuclear (PN) mitochondria.

38 These unique mitochondrial subsets are suggested to be physiologically and morphologically different (165); however, their morphological characteristics have not been comprehensively studied in the adult heart.

1.4.1 Mitochondrial Subtypes: IMF and SSM Mitochondria

In adult cardiomyocytes, IMF and SSM mitochondria exhibit a heterogeneous morphology and physiology (166; 167). IMF mitochondria from cat ventricular papillary and atrial muscle are 2 to 3 µm in length and around 0.1 µm in diameter (168;

169). Comparatively, the length of cardiac Japanese monkey (Macaca fuscata) IMF mitochondria are about 1.5 to 2 µm long whereas SSM mitochondria from the same animal measures from 0.4 to 3.0 µm in length (170). The morphological differences in these two mitochondrial subtypes are also present in skeletal muscle where IMF mitochondria are longer and less circular than the SSM mitochondria (171).

Conversely, IMF mitochondria from human sural quadriceps muscle have 64% smaller area when compared to the SSM mitochondria (172) thereby suggesting species- specific differences in mitochondrial morphology. Nevertheless, careful examination of the literature reveals that the investigation regarding the mitochondria morphology is often limited to the crude measures of density or mitochondrial area. These parameters cannot comprehensively explain the nature of shape changes associated with mitochondrial morphology under physiological and pathophysiological scenarios, and hence are inconclusive.

The morphological differences including size and length allow cardiac IMF to have a larger Ca2+ capacity in comparison to the SSM (173). In line with this fact,

Palmer et al. (1986) showed that after Ca2+ uptake assay, isolated rat SSM

39 mitochondria became dysmorphic, lost their IMM integrity and released their cytochrome C. In contrast, IMF mitochondria exhibited very subtle disorganisation of

IMM and did not release cytochrome C or any other enzyme markers when subjected to the same level of Ca2+ (174). Similarly, porcine cardiac IMF mitochondria also exhibit higher complex-2 induced Ca2+ retention compared to their SSM counterpart

(175). Taken together, the morphological characteristics of IMF mitochondria allow them to be more efficient in terms of energy production for cardiac contractility.

Some of the underlying differences between these two subsets can be also linked to their cristae organisation. The cardiac IMF mitochondria from male albino rats have marginally increased cristae length in comparison to their SSM counterpart which may be due to the larger size of IMF mitochondria (176). IMF mitochondria have mostly extended and tubular cristae whereas SSM mitochondria possess largely fenestrated and lamelliform cristae in Sprague-Dawley rats (166). In comparison to these two strains of rat, IMF mitochondria from Male Fischer 344 rats have a similar level of lamelliform cristae which indicates a heterogeneous mitochondrial structure across different strains of the same species (177). These observed morphological phenotypes highly correspond to the physiology of both cardiac SSM and IMF mitochondria (165). Following on from this, enzymatic activity, as well as substrate uptake for oxidative phosphorylation, are faster in IMF mitochondria than SSM mitochondria (178). IMF mitochondria also have greater state 3 respiration, exhibiting higher expression level of complex II and IV of the electron transport chain in comparison to the SSM (179). In addition, in the presence of the same respiratory substrates, IMF mitochondria display higher superoxide formation in comparison to the SSM (180).

40 Furthermore, the structural and physiological differences between these two subpopulations of cardiac mitochondria have been suggested, in part, to be due to the differences in their protein content (181; 182). In general, IMF mitochondria have a slower rate of protein synthesis including the respiratory chain enzymes when compared to the SSM mitochondria (183). SSM mitochondria have higher levels of

Opa1 in comparison to the IMF mitochondria (181). Prominent levels of Connexin-

43 (Cx43) are also expressed by the SSM mitochondria, whereas it is absent in IMF mitochondria (182). Cx43 participates in ATP production and oxidative respiration of

SSM mitochondria. It is localised to the IMM and its downregulation induces mitochondrial fragmentation and cytochrome C release suggesting that it has a role in mitochondrial dynamics (184; 185). Therefore, it is tempting to speculate that the difference in protein expression affects the extent of fission and fusion of these mitochondrial subtypes, which in turn influences the mitochondrial respiration and physiology as well as their dynamics.

The morphology and physiology of IMF and SSM subpopulations of cardiac mitochondria are often altered in different cardiac pathologies. For example, conditions that are associated with low oxygen reserves such as ischaemia affect cardiac IMF and SSM mitochondria by altering their shape and physiology (186).

Under hypoxic conditions, cardiac IMF and SSM mitochondrial density and cristae integrity are markedly reduced (176). Similarly, 45 minutes of ischaemia in the rabbit heart induces the formation of abnormal mitochondrial vacuolation and cristae deformity in both IMF and SSM mitochondria (186). The morphological changes in different cardiac mitochondrial subtypes under ischaemic conditions are coupled to their functional alterations (187). Acute IRI induces greater mitochondrial swelling in

SSM than IMF mitochondria in the presence of succinate as a respiratory substrate. In

41 addition, SSM mitochondria do not benefit from ischaemic preconditioning in the absence of the same respiratory substrates, and exhibit marked swelling and depolarisation (187). Despite being insensitive to IPC, SSM mitochondria are more responsive to the protection elicited by diazoxide in the setting of Ca2+ overload (188) which indicates the subtype-related response to cardioprotective treatments. It is noteworthy that ischaemia depletes the cardiolipin content of SSM mitochondria

(189), which can potentially impact the mitochondrial fission and fusion dynamics of this mitochondrial subtype and provide an explanation for the observed morphological and physiological changes.

1.4.2 Mitochondrial Subtypes: PN Mitochondria

Research surrounding the physiology and morphology of PN mitochondria is somewhat incomplete. PN mitochondria are tightly packed around the nucleus of cardiomyocytes and participate in nuclear import and function (190). PN mitochondria are generally smaller and rounder in comparison to the other two subtypes of adult cardiac mitochondria (170). However, the number of cristae and their cristae length do not substantially differ from the other mitochondrial subpopulations (176). PN mitochondria also respond to pathological stimuli in a comparable manner to the other two subtypes of cardiac mitochondria. They shrink in size in response to hypoxia and lose their cristae length and integrity (176). An investigation in porcine cardiomyocytes following MI has shown that the PN and SSM mitochondria which were positioned adjacent to the MI zone had a smaller area and were more circular.

Comparatively, the same paper documented the absence of any effect on IMF mitochondria post-MI (191).

42 Furthermore, the PN mitochondria are also functionally unique. Intact PN mitochondria of cardiomyocytes obtained from rabbit heart have lower resting Ca2+ and isopropanol-induced Ca2+ peak in comparison to their IMF counterparts (192), perhaps because of their smaller size. In addition, PN mitochondria exhibit lower levels of Ca2+ sparks in comparison to the IMF mitochondria (193). An elegant study by Cheng’s group showed that adult cardiac PN mitochondria haven nearly three times faster fusion rates than the IMF mitochondria (97). However, the reason behind this marked difference was not explained. Considering the fact that mitochondrial calcium handling is directly proportional to the mitochondrial content exchange in the form of direct fusion events and nanotunneling (89; 194), a different mechanism independent of Ca2+ signalling seems to be regulating the higher fusion activity of PN mitochondria.

Taken together, PN mitochondria exhibit a similar degree of association between their unique morphology and physiology when compared to the SSM and IMF mitochondria.

The notion of the existence of mitochondrial subpopulations was recently challenged by Rassaf et al. (2017). They argued that mitochondrial subpopulations are randomly absent in 2D images and are overall interconnected throughout the cell

(195). A possible explanation for the first argument is that the authors failed to pay specific attention to the fact that cardiomyocytes and mitochondria themselves are 3D entities and hence an absence of mitochondrial subtypes may be solely due to their spatial organisation (196; 197). The latter case can be addressed by emphasising the necessity for a comprehensive assessment of mitochondrial shape descriptors of individual mitochondrial subtypes. As such, in their article authors used only mitochondrial area and density parameters to identify different subsets of adult cardiac mitochondria (171). Furthermore, lack of clarification in terms of biological replicates

43 and the usage of cardiomyocytes with pathological morphology may have biased the inferences made by the authors to challenge the current view concerned with the existence of different mitochondrial subpopulations.

Mitochondrial Dynamics in the Adult Cardiomyocytes: KO Mouse Models of

Proteins That Participate in Mitochondrial Dynamics

The direct evaluation of mitochondrial morphology remains challenging due to technical difficulties associated with their in vivo examination within the intact cardiomyocyte. In order to overcome this challenge, several laboratories have developed indirect assessment methods that depend on modifying the gene (s) of interest that participate in mitochondrial dynamics in either an acute or chronic manner

(198–205). The use of cardiac-specific or whole-body modification of genes that are involved in the mitochondrial morphology and their impact on normal cardiac development and physiology will be the focus of the next sections.

1.5.1 Animal Models of Proteins That Participate in Mitochondrial Fusion

1.5.1.1 Cardiac-Specific Mfn1 and Mfn2 KO Mice

Mitofusins are active participants of mitochondrial fusion and influence differentiation and growth of cardiomyocytes (200; 201). Early studies using Mfn1 or

Mfn2 whole body KO mice showed that mice lacking these proteins die in mid- gestation (30). MEFs derived from the Mfn1 or Mfn2 mice have a fragmented network of mitochondria and lack a balanced fusion activity (30). Similarly, Mfn1 and Mfn2 double KO (Mfn1/2 DKO) mice die because of HF at the age of 7 days postnatally.

Mitochondria derived from Mfn1/2 DKO mice have a spherical phenotype and display marked mtDNA loss (200). In parallel, specific cardiac deletion of both mitofusins is

44 associated with abrogated mitochondrial morphology and respiration which ultimately results in lethal dilated cardiomyopathy at 8 to 9 weeks (198). Taken together, these studies indicate the significance of mitofusin function in cardiac embryonic development.

A careful review of the literature shows that some of the detrimental effects seen in Mfn1/2 DKO mice are solely due to the absence of Mfn2. It is known that the ablation of Mfn2 in mouse embryonic stem cells (ESCs) triggers Notch1 hyperactivity that leads to the differentiation arrest of ESCs. The differentiation arrest in Mfn2 deficient ESCs is dependent on the function of calcineurin Ca2+ signalling since pharmacological inhibition of calcineurin via FK506 can restore the differentiation process (206). Mitochondria from perinatal cardiomyocytes that express non- phosphorylatable mutant Mfn2 are unable to undergo mitophagic culling by Parkin and ultimately remain immature and unfit for metabolism. The adult hearts from these non-phosphorylatable mutant Mfn2 mice exhibit a glycolytic orientated profile rather than fatty acid derived metabolism which further underlines that the proper functioning of Mfn2 is crucial for cardiomyocytes differentiation (152). In contrast to cardiac specific Mfn2 KO hearts, 18-weeks-old Mfn1 cardiac specific KO mice exhibit normal left ventricular function with the absence of any cardiac hypertrophy. Despite these normal physiological phenotypes, cardiomyocytes from these mice show an extensive mitochondrial fragmentation and display a 40 % increase in the number of mitochondria versus their wild type (WT) counterpart (207). These results suggest that the ablation of Mfn2 is associated with more severe cardiac phenotype in comparison to Mfn1 KO. These effects can be attributed to the pleiotropic function of MFN2 in mitophagy and its role in facilitating mitochondrial cross-talk with the sarcoplasmic reticulum (SR) (152; 199).

45 The detrimental impacts of mitofusin deficiency are not limited to cardiac differentiation and growth. For instance, Mfn1 KO hearts exhibit abrupt expression of apoptotic and mtDNA regulators (207). Comparatively, cardiac-specific deletion of

Mfn2 induces cardiomyopathy; enlargement of mitochondria that result in compromised mitochondrial metabolism; SR-mitochondria dissociation leading to aberrant bioenergetics regulation; inhibition of autophagy and accumulation of autophagosomes-lysosome that leads to cardiomyopathy and mtDNA deficiency with the consequence of abolished cardiac mitophagy (199; 201; 208–210) which further unravels that cardiac homeostasis would be defective in the absence of mitofusins.

1.5.1.2 Opa1 in Adult Murine Heart

With its primary roles in IMM fusion and mitochondrial cristae regulation,

Opa1 acts as a critical mediator of mitochondrial metabolism and development (211).

Whole body Opa1 KO mice die in utero at day 9. Zebrafish model of Opa1 KO exhibits substantial oedema, dysmorphic atria, reduced circulation and reduced cardiac contractility (211). In addition, mutation of the Opa1 gene in ESCs induces growth arrest and inhibition of cardiac contractility (206). In comparison to the homozygous

Opa1 KO mice, 6-months-old heterozygous Opa1+/- KO mice have a normal cardiac function but exhibit enlarged mitochondria with deformed cristae (202). Likewise, twelve-month-old Opa1+/- KO mice exhibit marked mitochondrial fragmentation, disintegrated cristae, and reduced mtDNA copy number. In contrast to 6-months-old mice, aged Opa1+/- mice show a reduction in cardiomyocytes calcium transients leading to abnormal cardiac contractility as well as decreased cardiac output (212).

Therefore, the absence of Opa1 can lead to physiological dysfunction and developmental deficit.

46 1.5.1.3 Other Components of Mitochondrial fusion

Other members of mitochondrial fusion including cardiolipin also contribute to mitochondrial function and development in cardiomyocytes. Cardiolipin levels are reduced in human Barth syndrome by a mutation in the Tafazzin gene, which helps in the structural maturation of cardiolipin (213; 214). The reduction in cardiolipin levels is suggested to contribute to the reduction of mtDNA copy numbers, as well as abnormal alteration of mitochondrial morphology, which ultimately contributes to the cardiomyopathy seen in the Tafazzin deficient mice (213). Having stated the above, the direct evidence of the contribution of Cardiolipin and another member of the mitochondrial fusion proteins, MTIOPLD, in the context of cardiac homeostasis is limited.

1.5.2 Animal Models of Proteins That Participate in Mitochondrial Fission

1.5.2.1 Drp1 KO and Cardiac Phenotype

Mitochondrial fission is also responsible for cardiac growth and metabolism.

During early development, myoblasts rely on the fission activity of both Drp1 and mitophagic proteins for the distribution of their mitochondria, which eventually allows their transformation into a myotube (215). Mouse models of the whole body homozygous KO of Drp1 protein suffer from heart defects at E 10.5 and die in utero

(157). Furthermore, muscle-specific genetic ablation of Drp1 in mice disintegrates mtDNA and subsequently induces an elevation of α-actinin expression as well as myofibrillar disorganisation which follows a severe cardiomyopathy at postnatal day

7 (216). Besides, Drp1 KO hearts have disrupted mitophagy which contributes to the development of their cardiomyopathy. In addition, mitochondria from Drp1 KO

47 cardiomyocytes are abnormally enlarged and deficient in O2 consumption (216).

Likewise, cardiac-specific deletion of Drp1 mediates the accumulation of enlarged mitochondria that have decreased activity of complex I, II and IV. On the postnatal day 11, cardiac-specific Drp1 KO mice have marked reduction of fractional shortening and show a modest level of hypertrophy due to an upregulated autophagy in a parkin- independent (204) and MPTP-dependant (26) manner. Hence, embryonic development and normal cardiac physiology do not solely rely on mitochondrial fusion and require the action of mitochondrial fission proteins.

1.5.2.2 Other Components of Mitochondrial Fission

Other members of mitochondrial fission including Mff are known to be essential for normal functioning and development of mammalian hearts (205; 217).

Intriguingly, Mff mutation has been shown to cause mitochondrial-related- encephalomyopathy in human patients (217). Mice carrying whole body Mff ablation die at the age of 13 weeks. Mitochondria from Mff KO mice do not show abrupt morphological changes; however, they exhibit a marked reduction of oxygen consumption rate and respiratory enzymes. This ultimately leads to the development of cardiomyopathy followed by HF (205). The evidence regarding other members of mitochondrial fission such as hFis1 and MiD49/51 in altering the cardiac function is lacking. While much attention has been paid to the impact caused by the ablation of mitochondrial dynamics proteins on cardiac tissue and mitochondrial shape, it is currently unknown whether their absence alters the actual occurrence and rate of fission and fusion in the adult heart.

48 Mitochondrial Dynamics in Health and Diseases

Upregulation or downregulation of fission and fusion events in the heart have been implicated in a number of CVDs. The relevance of mitochondrial dynamics on cardiovascular-related pathophysiology will be briefly reviewed in the following sections.

1.6.1 Heart Failure (HF)

The impairment of mitochondrial fission/fusion balance has been suggested to be one of the main hallmarks of HF (218). The pathophysiology of HF is associated with a substantial reduction of Opa1 level which results in a marked decrease in mitochondria area and an increase in mitochondrial population (219). YME1L KO mice have an upregulated expression of the OMA1 protein which breaks down L-

Opa1. Cardiomyocytes from YME1L KO mice have extensive mitochondrial fragmentation and gradually exhibit a shift from β-oxidation to glycolytic metabolism that ultimately leads to HF (220). Mitofusin deficiency is also associated with HF. In the absence of mitofusins, mitochondria from tamoxifen-induced cardiac-specific

Mfn1/2 DKO hearts lose their fusion balance. This process results in the reduction of up to 40% of the mitochondrial area due to the abundance of mitochondrial fission.

Mfn1/2 DKO mice eventually develop HF at the age of 7-8 weeks post-Mfn1/2 ablation (198). In comparison to fusion proteins, the genetic ablation of Mff has been reported to reduce mitochondrial density and disturb respiration to trigger cardiomyopathy and HF. It is noteworthy to mention that the same authors reported that the heterozygous ablation of Mfn1 reverses the phenotypes in Mff KO mice suggesting the importance of balanced fission and fusion for normal functioning of the myocardium (205). Apart from Mff, loss of Drp1 in the heart also stimulates the formation of elongated mitochondria that are more susceptible to MPTP opening. Mice

49 with this defect develop HF after 6-7 weeks post Drp1-ablation (26; 221). In addition, down-regulation of Drp1 abrogates the process of autophagy in mice undergoing transverse aortic constriction and augments mitochondrial dysfunction before triggering HF (222). Although the therapeutic targeting of mitochondrial fission by mitochondria division inhibitor-1 (mdivi-1) has been shown to be beneficial in terms of pressure-overload induced HF (223), restoring the equilibrium between fission and fusion should be the ultimate goal for the treatment of HF.

1.6.2 Pulmonary Arterial Hypertension (PAH)

PAH is a cardiopulmonary disease which is known for its heterogeneous pathophysiology which mostly involves the upregulation of mitochondrial fission

(224). Pulmonary artery smooth muscle cells from patients with PAH have an elevated level of fragmented mitochondria. The increased fragmentation is mainly due to the increased hFis1 level as well as the elevated phosphorylation of Drp1 at S616 domain and downregulation of Mfn2 protein (10; 225). PAH mediates an increase in the level of microRNA-140 which negatively regulates the expression of Mfn1 (226).

Ischaemia is also known to induce exacerbated upregulation of mitochondrial fission components including Drp1 and hFis1 in the presence of PAH (227). Likewise, the transformation of contractile rat aortic smooth muscle cells to a hyperproliferative phenotype in the setting of early development of PAH occurs due to the downregulation of Mfn2 (228). In contrary, the proliferation of pulmonary artery smooth muscle cells has been also documented to require Mfn2 upregulation (229).

Moreover, Cessation of mitochondrial fission either by inhibition of Drp1 and hFis1 via Mdivi-1 and P110 or by overexpression of Mfn2 has been shown to reverse the detrimental effect of PAH (10; 227; 228; 230). Therefore, fission rather than fusion is crucial for the induction of PAH.

50 1.6.3 Diabetic Mellitus (DM)

Although the direct involvement of mitochondrial fission and fusion in the pathophysiology of complications associated with diabetic hearts are not fully understood, the current findings allude to the prominent role of mitochondrial fission in the aetiology of DM (231; 232). Hyperglycaemia is one of the hallmarks of DM.

Hyperglycaemic treatment of H9C2 myoblasts is correlated with increased ROS production and Drp1-induced mitochondrial fragmentation. Hyperglycaemia also shifts the mitochondrial distribution towards the nucleus and decreases both the form factor (a measure of perimeter) and aspect ratio (length-to-width ratio) of individual mitochondria (233). Drp1 inhibition by Mdivi-1 has been shown to protect primary neonatal rat cardiomyocyte from hypoxia reoxygenation (HR)-induced cell death in a hyperglycaemic condition (234). Similarly, Mdivi-1 treatment of high-fat diet and streptozotocin-induced diabetic mice has been shown to lower MI size after acute IRI

(235). It is noteworthy to also acknowledge that the anti-diabetic drug, metformin, suppresses the DM associated atherosclerosis formation by preventing mitochondria fragmentation via suppression of Drp1 (236) which additionally implicates the role of

Drp1 in the progression of DM.

The role of fission in DM becomes more evident when we study the alterations of mitochondrial fusion proteins in DM. Hearts from diabetic (db/db, leptin receptor- deficient) mice possess hyperacetylated Opa1, which has lost its GTPase activity to induce fusion (70). Likewise, endothelial cells derived from streptozotocin-induced diabetic Sprague Dawley rats have increased ROS production and display an increased level of mitochondrial fragmentation due to the downregulation of Opa1 and a surge in Drp1 levels (237). Conversely, a recent investigation has shown that skeletal- muscle-specific ablation of Opa1 can rescue the whole-body insulin resistance

51 associated with DM (238). Therefore, these findings indicate the tissue-specific pleiotropic role of Opa1 in the pathogenesis of DM. Furthermore, mitofusins are also affected by the DM since the continuous development of diabetes in obese rats reduces

Mfn2 expression profile (239). Mitochondria from the right atrial tissue of type 2 diabetic patients undergoing cardiopulmonary bypass surgery appear mostly fragmented, mainly due to the reduction of Mfn1 protein. The alteration of shape in these mitochondria is accompanied by the reduction in the expression of respiratory complexes I, II and III. (13). Similarly, mitochondria from high fat diet-related diabetic mice have lower mitochondrial fusion events due to decreased Mfn1 and Mfn2 expression as well as increased Drp1 and hFis1 level (240). Taken together, the main component of mitochondrial dynamics including mitochondrial morphology as well as mitochondrial fusion dynamics are impaired due to excessive fission in DM.

Nevertheless, there is insufficient evidence to show the exact alteration of mitochondrial fission and fusion proteins in both types of DM and hence more research is essential to explore the exact role of the mitochondrial dynamics in the pathogenesis and therapeutics of DM.

1.6.4 Ischaemia Reperfusion Injury

Following acute IRI a cascade of events including the rise in intracellular Ca2+, pH alterations and surge in ROS generation, results in MPTP opening and cell death

(241). These events influence both members of mitochondrial fission and fusion and drastically alter mitochondrial dynamics (242). The induction of HR in H9C2 cardiomyoblast cell lines mediates mitochondrial swelling as well as a drop in the membrane potential which in turn initiates the formation of doughnut- shaped/fragmented mitochondria. These events require the action of the Drp1 protein.

HR also reduces mitochondrial fusion events and disturbs the balance of mitochondrial

52 dynamics (92). The modulation of mitochondrial morphology by inhibiting mitochondrial fission inducer, Drp1, has been shown to offer protection against lethal acute IRI (7–9) but whether it can also rescue the occurrence of fusion is not known.

In both in vivo and ex vivo models, inhibition of Drp1 by Mdivi-1 reduces MPTP opening and MI size in rat hearts following acute IRI (9; 17). In vivo pre-treatment of rat hearts with a selective inhibitor of hFis1/Drp1, P110, prevents the loss of mitochondria area, by inhibiting mitochondrial fragmentation, and decreases MI size by 28% (8). In parallel, homozygous Mff KO mice have reduced mitochondrial fragmentation, MPTP opening and MI size after microcirculatory IRI insult (243).

These studies have contributed to the rise of the notion that mitochondrial fission may be ‘bad’ for cardiac function under pathophysiological conditions. However, investigations using mice deficient in Drp1 have suggested that chronic inhibition of mitochondrial fission is detrimental. Ikeda et al. (2014) found that the cardiac-specific

Drp1 KO mice have an increased susceptibility to apoptosis and have a raised level of

MPTP opening. The same authors also reported that the Drp1 KO hearts have a greater

MI size after acute IRI in comparison to the WT mice (156). These facts clearly reveal the significance of Drp1 function in the heart and distinctly indicate that the acute therapeutic benefits of Drp1 inhibition are lost in chronic genetic KO models.

In terms of mitochondrial fusion components, deficiency in mitofusins have been widely studied in the setting of cardioprotection following IRI (199; 201; 208;

244). Our group and others have shown that mitochondria from Mfn1/2 DKO hearts have surprisingly a lower MI size in comparison to their WT counterpart (198; 244).

The cardiomyocytes from these hearts have reduced mitochondrial area and exhibit diminished state 3 respiration (198; 244). The protection seems to be dependent on both Mfn1 and Mfn2. Mitochondria from single Mfn1 cardiac specific KO hearts are

53 smaller than their WT counterpart and have reduced Ca2+ uptake capacity which protects them from ROS-induced MPTP and cell death (207). However and contradictory to this fact, MI size in hearts deficient in a negative regulator of Mfn1, miRNA 140, has been shown to be smaller (140). In comparison to Mfn1, the role of

Mfn2 in cardioprotection seems to be more complex (16). Apart from its role in mitochondrial fusion, Mfn2 also tethers mitochondria to the SR and synchronises cardiomyocyte Ca2+ handling and contraction (199; 245). The loss of tethering in

Mfn2-deficient cardiac mitochondria induces lower mitochondrial Ca2+ uptake (199), however, whether this reduction is due to the anatomical alteration or other pleiotropic roles of Mfn2 has remained elusive. In addition, mitochondria from Mfn2 KO hearts are enlarged, have higher Ca2+ retention capacity and show delayed depolarisation when subjected to ROS-induced stress. These characteristics together contribute to the lower HR -induced cardiomyocytes death and reduced MI size in Mfn2 KO hearts in comparison to WT hearts (201; 246). While ablation of Mfn2 in the heart can mediate protection, overexpression of Mfn1 or Mfn2 in HL-1 cells can induce elongation and reduce the MPTP related cell death (7). In parallel, in vivo overexpression of Mfn2 protein prevents atherosclerosis formation by preventing vascular smooth muscle cells proliferation via inhibition of AKT and ERK (247). Overall, with the inhibition of mitochondrial fusion demonstrating cardio-protection, the mantra that fusion is good, and fission is bad may be too simple and underlines why more work is required to fully understand the role of these proteins in terms of fusion/fission, and their other roles in

IRI.

Having previously discussed the structural alterations associated with Opa1 mutations, the expression level of Opa1 is also significantly altered during acute IRI

(16). During ischaemia and in the presence of H2O2 induced oxidative stress, L-Opa1

54 is rapidly proteolysed into S-Opa1 by the action of OMA1. Subsequently, the reduction of L-Opa1 leads to the induction of mitochondrial fragmentation which is accompanied by the release of cytochrome C and cell death (248; 249). Methods to rescue the proteolytic cleavage of the L-Opa1 level by its in vivo overexpression can reduce MI size following IRI (250). On the other hand, 12-months-old Opa1+/- mice isolated cardiomyocytes treated with 6 hours of hypoxia and 1 hour of reoxygenation display a significantly higher cell death in comparison to WT cardiomyocytes (212).

In line with this data, 3 months old Opa1+/- hearts subjected to 30 minutes ischaemia and 2 hours of reperfusion had higher MI size in comparison to the WT. Interestingly, the same study also found that isolated cardiac myocytes had the same level of mPTP but the mitochondrial Ca2+ uptake was reduced in the Opa+/- mice (251). These findings conflict with the work of Piquereau et al. (2012) where they documented higher Ca2+ uptake and delayed mPTP in Opa+/- mice (202). These contrasting effects of Opa1 versus mitofusins in the setting of cardioprotection add further complexity to the therapeutic targeting of mitochondrial fusion proteins in the setting of IRI.

One of the main therapeutic approaches for targeting IRI are short cycles of ischaemia and reperfusion given prior to the ischaemic insult and is termed “ischaemic preconditioning (IPC)”(241). The exact mechanism by which IPC influences mitochondrial dynamics following IRI is inconclusive. Remote IPC in male adult

Wister rats, given prior to 40 minutes of ischaemia and 120 minutes of reperfusion, has been shown to increase Opa1 expression and reduce MI size. The same study also reported that IPC had no effect on Drp1 expression (252). In comparison, pharmacological preconditioning using penehyclidine hydrochloride given to Wister rats that were subjected to 30 minutes of ischaemia and 3 hours of reperfusion were shown to reduce MI size by inhibiting Drp1 expression and stabilising Mfn1 and Mfn2

55 levels (253). Similarly, pharmacological nitrate treatment of ex vivo Langendorff hearts from male Sprague–Dawley rats, subjected to 20 minutes of ischaemia and 2 hours of reperfusion inhibited Drp1 phosphorylation at S656 domain via the action of

PKA (254). However, despite comprehensive investigations about proteins involved in mitochondria dynamics, the nature of morphological and communicational changes in the setting of IRI and cardioprotection against IRI remains poorly understood.

Methods to Assess Mitochondrial Dynamics

The assessment of cardiac mitochondrial morphology and dynamics in the form of intra-mitochondrial communication has been achieved using either electron or light microscopy and the pros and cons of these methods will be reviewed in this section.

1.7.1 Electron Microscopy Assessment of Mitochondrial Morphology

2D electron microscopy has generally been used to assess mitochondrial morphology in fixed cardiac cells (254–256). Although the nature of morphological differences of mitochondrial subpopulations has been well studied in skeletal muscle using 2D electron microscopy (171; 258), their characteristics and importance to the adult cardiomyocyte are mostly limited to single measures of shape (198; 201) and hence are unknown.

In spite of offering a higher resolution of up to 0.2 nm to light microscopy for the assessment of mitochondrial shape, this technique often requires the dehydration of the fixed sample which affects both 2D and 3D morphology of the organelle (259).

Mitochondrial shape and network integrity analysis are often conducted in 2D and the measurement associated with their 3D shapes such as volumetric features are ignored.

56 This is mainly due to the lack of assays designed for 3D shape evaluation (197).

However, several techniques have been recently implemented to assess mitochondrial structure in 3D including serial thin section tomography (STS), focused ion beam

(FIB) tomography and serial block face scanning electron microscopy (SBF-SEM)

(260). 3D STS of hearts from Wister rats have been used to show the 3D structural heterogeneity of cardiac mitochondria in terms of Ca2+ signalling (260). Volumetric data obtained using FIB was used by Rennie et al. (2014) to reconstruct chicken heart outflow and show that around 45% of the embryonic tract is comprised of mitochondria (261). Using sheep and rat hearts analysed by SBF-SEM, Pinali et al.

(2014) showed that the 3D structure of the t-tubule-mitochondria-SR network is substantially diminished after HF (196; 262). Whilst more attention has been paid to the volumetric analysis for the 3D structure of the cardiac mitochondrial network, less is known about the 3D structural differences of individual mitochondria in the adult heart.

1.7.2 Light microscopy

Light microscopy remains the technique of choice to assess mitochondrial dynamics in living cells. Light microscopy often accompanies the use of positively charged fluorescent probes to stain mitochondria based on their negative membrane potential (263). Mitochondrial dynamics and function in mammalian cells such as cardiac myocytes can then be investigated using the fluorescent characteristics of individual probes (264; 265). Most of the membrane potential dyes including TMRM,

JC-1, Rhod-123, DIOC and MitoTracker have varying degrees of mitochondrial cytotoxicity since they disturb mitochondrial respiration and induce free radical formation (263; 266). At high concentrations, they can also leak into the cytosol thereby disturbing the image quantification process (266). Green fluorescence protein

57 (GFP) offers higher brightness than the conventional membrane potential dyes and can be designed for genetic expression along with the mitochondrial protein of interest.

However, these proteins can sometimes interfere with the normal function of the protein of interest or are expressed in high quantity and become cytotoxic (266).

Genetically targeted mitochondrial proteins are not membrane potential based and hence are advantageous for studying mitochondrial morphology in experiments where the membrane potential is affected. In addition, genetically modified mice expressing fluorescence proteins can allow in vivo quantification of mitochondrial morphology without the complications mentioned above (267; 268).

Actual imaging and quantification of cardiac mitochondria by using light microscopy, are often restricted to the semi-quantitative and crude measures of shape

(190; 207; 269; 270). The morphology of cardiac mitochondria can be assessed using

FACS, but this method requires mitochondrial isolation and has lower resolution in comparison to confocal microscopy (271). Comparatively, confocal microscopy offers higher resolution than FACS and allows in vivo assessment of mitochondria in intact cells. However, imaging cardiac mitochondria using confocal microscopy is also restricted due to the out of focus light and the limited axial resolution which is associated with thicker cells such as cardiac cells (272). Despite these facts, the out of focus light and image resolution can be restored by the process of deconvolution that may allow precise quantification of individual cardiac mitochondria using the confocal microscopy (273). The lack of resolution may be also compensated by the use of super- resolution microscopy (SRM), however, current application of SRM for thicker cells are limited due to the associated technical difficulties such as photo-bleaching and cumbersome computational image processing for 3D volumes (274). Overall, the

58 enhancement of light microscopy techniques seems crucial to enable more detailed assessment of the mitochondrial morphology in adult cardiomyocytes.

Mitochondrial dynamics in the form of fission has not been documented in the adult heart and the process of fusion is also poorly understood but is beginning to emerge (89; 97; 231). Primary studies by Hajnóczky et al. (2011) showed that mitochondrial fusion events in H9C2 cardio-myoblasts can be tracked using two colour PAGFP and DSRED (92). The same group and others also used the viral transfection of two or single colour photoactivatable GFP to show the occurrence of fusion events in adult murine hearts (89; 97). Mitochondrial dynamics can be also studied using single colour fluorescence recovery after photobleaching; however, mitochondrial motility can affect the interpretation of the results (275). Taken together, while the assessment of mitochondrial fusion in adult cardiac cells can be achieved using photoactivable probes or photobleaching, the direct detection of mitochondrial fission is more challenging in the adult cardiac cells and requires the development of new techniques.

59 Chapter 2. Overall Aims and Objectives

Main Hypothesis

Despite the wealth of knowledge covering the alteration of mitochondrial proteins that govern the organelle dynamics, mitochondrial dynamics itself has been rarely studied in adult cardiac myocytes. This is due to the lack of imaging techniques available for quantitative assessment of both morphology and communication of adult heart mitochondria in vivo and thus the significance of these characteristics to the adult cardiomyocytes remains unclear. Hence, we hypothesised that the changes in mitochondrial dynamics in terms of shape characteristics and inter-mitochondrial communication in the form of fusion events are important and relevant to adult cardiac myocytes.

Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of

Mitochondrial Morphology in Adult Cardiomyocytes

2.1.1 Rationale:

The morphological characteristics of adult cardiac mitochondria, which are divided into three disparate subpopulations (IMF, PN and SSM), can be studied using conventional 2D transmission electron microscopy (TEM) in vivo (198; 201).

However, the 2D assessment of cardiac mitochondrial morphology is often limited to a few parameters of shape such as “mitochondrial area” which cannot comprehensively allow the exact determination of mitochondrial shape differences.

Besides, whether the shape parameters of adult cardiac mitochondria alter under pathophysiological conditions is not clear.

60 2.1.2 Aims and Objectives:

The main objectives were to quantify the 2D morphology of adult cardiac mitochondria under normal and pathophysiological conditions and included:

❖ To characterise the morphological shape parameters of three distinct

subpopulations of adult mouse cardiac mitochondria using 2D TEM.

❖ To assess the effect of ischaemia and IPC on adult cardiac mitochondrial 2D

shape descriptors using the 2D TEM.

❖ To determine the effect of ablation of both Mfn1 and Mfn2 on adult cardiac

mitochondria by using the 2D TEM.

Quantification of Mitochondrial Dynamics in 3D: Shape Descriptors of

Mitochondrial Morphology in 3D

2.2.1 Rationale:

The shape descriptors of adult heart mitochondria are not restricted to the 2D plane. Similar to the cardiomyocytes themselves, mitochondria also exist in three planes and hence the 3D evaluation of cardiac mitochondrial shape can increase the sensitivity of 2D findings (197). Whether the 3D shape parameters of mitochondria are relevant to the adult cardiac myocytes have not been thoroughly investigated.

2.2.2 Aims and Objectives:

❖ To study the 3D shape descriptors of adult cardiac mitochondria by using WT

and MFN2 deficient cardiac myocytes.

❖ To assess the utility of specialised 3D electron microscopy techniques such as

FIB-SEM and SBF-SEM for evaluating the morphology of adult mouse cardiac

mitochondria

61 High-throughput Screening of Small Molecule Modulators of Mitochondrial

Dynamics

2.3.1 Rationale:

The importance of mitochondrial dynamics becomes more evident when we look at the studies targeting mitochondrial dynamics for cardioprotection in the adult heart. Our group and others have shown that the maintenance of fusion by acute cessation of mitochondrial fission, by targeting Drp1 protein, can eliminate the detrimental effects of IRI in the adult heart (7; 9). Hence, we hypothesised that there could be other modulators of mitochondrial dynamics and designed a high throughput screen to detect novel small molecule modulators of both mitochondrial fission and fusion.

2.3.2 Aims and Objectives:

The main objective was to use cell-based high-throughput screening to discover novel therapies for modulating mitochondrial morphology:

❖ To examine and characterise the use of an open source algorithm available in

image J to detect and measure mitochondrial shape descriptors in mtRFP

transfected HeLa cells

❖ To identify novel modulators of mitochondrial shape using the Prestwick

chemical library of small molecules

❖ To further evaluate the hits from the screen and select the potential candidates

to be tested in the setting of IRI and cardioprotection.

62 Mitochondrial Dynamics and Hydralazine-Induced Cardioprotection

2.4.1 Rationale:

Mitochondrial dynamics in the form of fusion events has been studied using either single or two colour imaging (92; 97) The physical occurrence of fission or fusion has not been fully documented in adult cardiomyocytes and in particular there is not much evidence to support the potential differences in fusion dynamics of different mitochondrial subpopulation of adult cardiac mitochondria under normal or pathological conditions (89). Using the high-throughput screen we identified hydralazine, as a novel inhibitor of mitochondrial fission. Considering that the acute inhibition of fission has been shown to elicit cardioprotection (7; 9) we hypothesised that hydralazine can protect the heart after IRI insult by rescuing mitochondrial fusion and inhibiting mitochondrial fission by affecting Drp1 function.

2.4.2 Aims and Objectives:

In this chapter, we sought to examine the mitochondrial fusion events in adult cardiomyocytes under normal and ischaemic conditions in the absence or presence of hydralazine by the objectives listed below:

❖ To characterise the fusion dynamics of three subpopulations of cardiac

mitochondria in mice expressing mitochondrial targeted Dendra2 under normal

and IRI conditions

❖ To investigate the effect of hydralazine on mitochondrial dynamics under

normal or IRI condition.

63 Chapter 3. Quantification of Mitochondrial Dynamics in 2D: Shape Descriptors of Mitochondria in Adult Cardiomyocyte

Introduction

Mitochondria exhibit heterogeneous morphology in mammalian cells under basal status. The difference in their morphology is highly dependent on their dynamic nature and physiological needs within cells (190). There are three distinct subpopulations of mitochondria in adult cardiac cells: IMF, SSM and PN (see Section

1.1 for a summary) mitochondria which possess diverse morphology and physiology

(165). IMF mitochondria are larger in size in comparison to the other two subtypes.

This feature helps IMF mitochondria sustain a greater respiratory function and increase their capability for Ca2+ handling (179; 192) to enable them to support contractility- required energy production. In comparison, PN mitochondria are suggested to be more efficient in nuclear import due to their smaller size and densely packed organisation

(190). Although the morphology of mitochondria has been shown to be fundamental to their function, the shape characteristics of the three subtypes of adult cardiac mitochondria have not been thoroughly studied.

The morphological characteristics of cardiac mitochondria alter under pathological conditions. Cardiac mitochondrial morphology is sensitive to pathological stimuli such as hypoxic and ischaemic conditions (22; 186). Hypoxic conditions have been shown to induce fragmentation of mitochondria in H9C2 cardiomyoblast cells and adult cardiomyocytes (22; 176). Similarly, ischaemia in canine myocardium has been shown to induce marked swelling and fragmentation of the different subpopulations of adult cardiac mitochondria (276). Moreover, therapies targeted against ischaemia such as IPC have been associated with signalling pathways that can downregulate the fission function of Drp1 (252–254), but whether IPC affects

64 the actual morphology of cardiac mitochondria is not known. Furthermore, how mitochondrial shape changes in response to ischaemia and whether IPC preserves mitochondrial shape after ischaemia is unknown.

Transcriptional expression of mitochondria fusion and fission proteins have been shown to affect the mitochondrial morphology in the heart (156; 198–201). Two previous studies have shown that the genetic ablation of both Mfn1 and Mfn2 can induce considerable mitochondrial fragmentation (198; 200). However, these studies did not comprehensively assess the extent and the type of morphological alterations in

Mfn1/2 deficient mice.

In this section, we characterised the morphological shape parameters of the three distinct subpopulations of adult mouse cardiac mitochondria using 2D TEM. We showed that these characteristics are altered after ischaemia and the cardioprotective

IPC treatment did not protect against the morphological changes after ischaemia.

Additionally, we further showed that some of these shape characteristics were influenced by the function of the mitofusin proteins.

Methods

3.2.1 Animals and Materials

All animal experiments were performed in compliance with the Animals

(Scientific Procedures) Act 1986 published by the UK Home Office. Male C57/BL6 mice aged 8-10 weeks old were used for the general characterisation sections. To study the effect of mitofusin deficiency on morphology of cardiac mitochondria, cardiomyocyte-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks using 5 days administration of tamoxifen (20mg/kg/day)(Myh6-

65 MerCreMer inducible model), and hearts were extracted at least 5 weeks after tamoxifen injection (198). MFN1,2 DKO mice were obtained by kind collaboration with Prof Gerald Dorn from Washington University. Genotyping was performed as explained in Section 4.2.2. All materials were purchased from Sigma unless otherwise stated.

3.2.2 Ex-vivo Langendorff Characterisation Using Infarct Size

Our initial aim was to characterise the Langendorff apparatus by assessing MI size following IRI and IPC. Mouse euthanasia was conducted using Sodium

Pentobarbitone (20mg/ml). After full euthanasia, hearts were harvested, cannulated, and perfused with Krebs-Henseleit buffer containing NaCl (118 mM), NaHCO3 (25 mM), d-Glucose (11 mM), KCl (4.7 mM), MgSO4.7H2O (1.22 mM), KH2PO4 (1.21 mM) and CaCl2.H2O (1.84 mM) using a constant pressure setup supplied with circulating 95% O2-5% CO2 at pH 7.35–7.45. Mouse hearts were randomly assigned to two separate experimental groups: a) Control group: Stabilisation (ST) for a total time of 55 minutes was followed by

35 minutes ischaemia and 90 minutes reperfusion.

b) IPC group: 25 minutes of ST was followed by 3 cycles of 5 minutes ischaemia/5

minutes reperfusion after which, hearts underwent global ischaemia for 35 minutes

before being subjected to 90 minutes reperfusion.

After each procedure, hearts were sectioned perpendicularly from apex to the base.

Each slice was then incubated with 1% triphenyltetrazolium chloride (TTC) for 15 minutes at room temperature to discriminate the infarct tissue (yellow area) from the viable myocardium (red area). The sections were scanned, and their images were

66 imported to Image J V.1.51. The infarcted area of individual sections was then segmented based on the signal intensity of individual regions as shown in Figure 3-1

A and B. Infarct size was expressed as % of the area at risk (the entire volume of left ventricle).

Figure 3-1 Delineation Example of Infarcted Area from Viable Myocardium in Control Group. (A) Shows the original images whereas (B) shows the segmented infarcted area and viable myocardium in yellow and red, respectively.

3.2.3 Tissue Fixation Characterisation and Processing for TEM

To assess the efficiency of fixation in terms of preservation of mitochondrial morphology and cell structure in normal C57/BL6 untreated male mice, three different methods of fixation were used including:

67 a) Group a: Hearts were extracted and perfused immediately with saline and were

then placed into the fixative solution (1% paraformaldehyde, 2% glutaraldehyde

in 0.1M sodium cacodylate buffer) (N=3).

b) Group b: After removal, hearts were perfused with saline and an area from the left

ventricle was cut and placed inside the fixative solution (N=3).

c) Group c: The harvested hearts were perfused with saline and were then

immediately perfused with the fixative (N=3).

Imaging was performed using the Joel TEM in a blinded fashion at 4K magnification.

Six random images were taken per heart and analysis was based on the crude measures of mitochondrial morphology or the presence of cell pathologies such as myocardial disarray and/or hypercontracture.

After selecting the most appropriate fixation method, hearts were fixed for at least 12 hours and a section from their left ventricle was subsequently dissected.

Samples were then further fixed in 2% osmium tetroxide, which stains lipid membranes, for 2 hours at 4°C. Individual sections were then dehydrated for 10 minutes in solutions containing different ethanol concentration (25%, 50%, 70%, 90%,

100%) and were washed for another 30 minutes (3x10 minutes) in 1,2-epoxypropane.

Samples were embedded in Epoxy resin (Poly/Bed-21844-1) and were oven dried for

48 hours. Ultrathin 70nm sections were obtained by ultra-microtome (Reichert) and stained using lead citrate.

68 3.2.4 Ex-vivo Langendorff Model for TEM

The characterisation of mitochondrial morphology under normal or ischaemic conditions in the presence or absence of IPC was performed using two individual

Langendorff rigs (N=6 hearts for each group). The main protocol is depicted in Figure

3-2 and involved the following groups:

Figure 3-2 Langendorff Ex-vivo protocol. This diagram illustrates the experiment protocol and the timing for each individual step within the protocol (Modified from (277)). 1. Stabilisation for a total time of 55 minutes (ST)

2. ST for a total time of 25 minutes followed by preconditioning regimen of three

periods of 5 minutes ischaemia/ 5 minutes reperfusion (ST+IPC)

3. ST for a total time of 55 minutes followed by 20 minutes of global ischaemia

(ST+IS)

4. ST for a total time of 25 minutes followed by IPC (30 minutes) before the induction

of global ischaemia for a period of 20 minutes (ST+IS).

69 3.2.5 Image Acquisition and Analysis

A Gatan Orius camera installed on Joel 101 TEM was used to capture images with 5 x 5 nm pixel dimensions. In each individual heart from different treatments, 6 disparate cells in their longitudinal plane were imaged blindly for every subpopulation of cardiac mitochondria. As shown in Figure 1-3, IMF mitochondria were defined as mitochondria that were present between the muscle fibers; SSM mitochondria were defined as mitochondria that were present exactly beneath the cell membrane; and PN mitochondria were defined as mitochondria filling the cavity in front of the cell nucleus often extending from the nucleus to the IMF or SSM regions. IMF mitochondria extending to the SSM regions or vice versa were excluded from the analysis. In total, 7,301 IMF mitochondria, 5,346 PN mitochondria and 3,739 SSM mitochondria were segmented and analysed. Cardiomyocytes which exhibited pathological ultrastructures such as loss of membrane, myocardial disarray, and hypercontracture were excluded from imaging. Z-line spacing was also assessed between groups to ensure cardiomyocytes with similar structure are analysed for each group. A single line was drawn between two adjacent Z-lines (covering the entire length of the cardiac myocytes) using the “straight line” tool of Image J software to assess the approximate sarcomeric length in different treatments. For each condition, three random cells from individual hearts were used to analyse the sarcomeric length.

For the MFN1/2 DKO sections (N=3), 6 cells in their longitudinal orientation covering the IMF mitochondria were blindly captured and mitochondria within those

6 cells (No. of Mitochondria WT: 1,145 and Mfn1/2 DKO: 1,212) were traced.

For image analysis, the following individual 2D morphometric parameters of cardiac mitochondria were obtained using Image J software (refer to image 3-2 and 3-3 for a visual explanation):

70 1. “Area in μm2”: corresponds to the surface occupied by individual

mitochondrial (Figure 3-3 B and 3-4 A-C);

2. “Perimeter in μm”; measures the outline length of the traced mitochondrion

(Figure 3-3 C and 3-4 A-C);

3. “Aspect ratio (major to minor axis ratio)”: where major and minor axis are the

longest and shortest line that can be drawn inside a fitted ellipse on the surface

of the traced mitochondrion (Figure 3-3 C (outline) and D (fitted ellipse) and

3-4 A-C);

4. “Feret’s diameter in μm: defined as the longest line that can be drawn between

any two points within a mitochondrion” (Figure 3-3 E and 3-4 A-C);

Figure 3-3 Visual Description of Mitochondrial Shape Parameters. Original EM micrograph is depicted in (A). The surface area and perimeter of the segmented mitochondrion are shown in (B) and (C). The major and minor axis of the fitted ellipse (based on the outline in (C)), which are used for calculating aspect ratio, are shown in (D) whereas E illustrates Feret’s diameter.

71 Apart from the above parameters we also assessed the measures of circularity and roundness to better understand the extent of mitochondrial sphericity and their deviation from an elongated shape (see the following formulas):

5. “Roundness (expressed as 4 x Area/π x Major axis2 or the inverse of aspect

ratio)”; relates to the mitochondrial sphericity; elongated mitochondria have a

longer major axis and hence have a lower roundness value (See Figure 3-4 A-

C).

Figure 3-4 Morphometric Evaluation of Mitochondria with Disparate Shapes. (A) depicts the original electron microscopy micrograph. The measurements of manually traced mitochondria in (B) are shown in (C).

6. “Circularity (expressed as 4π x Area/ Perimeter2)”. Mitochondria with

invaginations and elevated levels of branching, which possess longer

perimeter, have lower circularity value (See Figure 3-4 A-C).

72 3.2.6 Statistics

Statistical analysis was performed using either Graph Pad Prism or Stata (v.13).

The differences in infarct sizes were assessed using student t-test. The effects of the different fixation methods were assessed using the Kruskal-Wallis test. The mixed effects model was used to assess differences between the mitochondrial morphometric descriptors. The mixed effect included the random effect for incorporating the variability between cells and hearts of the hierarchical model as well as the fixed effect which consisted of subpopulations of mitochondria and different treatment groups.

Statistical modelling was performed in collaboration with Dr Qiao Fan and Dr Bibhas

Chakraborty (from Centre for Quantitative Medicine, DUKE-NUS). Data from mitochondrial area and aspect ratio were skewed and hence were logarithmically transformed. Similarly, the same test was used for the data from Mfn1/2 DKO hearts.

For the Mfn1/2 DKO dataset, measures of area, perimeter, Feret’s diameter and aspect ratio were logarithmically transformed. Graphs were made using either Microsoft

Excel 2010, Graph Pad Prism, or STATA. P value of ≤0.05 was considered significant.

Results

3.3.1 Characterisation of Langendorff Model Using the Infarct Size Assessment

Infarct sizes that are shown in Figure 3-5 A and B, clearly demonstrate that there was a marked reduction of infarct size in IPC group versus the control (Mean ±

S.E: 34.7% ± 1.6 vs. 10.9 % ± 0.7, P<0.0001). Therefore, we successfully demonstrated that IPC can protect against the detrimental effect of IRI.

73

Figure 3-5 Characterisation of Mouse Langendorff Model. Infarct sizes as a % of the left ventricular volume are depicted in A and representative images from control and ischaemic preconditioning (IPC) groups are shown in B.

*** denotes P<0.001 vs. control (N=6 per group).

3.3.2 Examination of Heart Fixation Method

Having demonstrated the protective effect of IPC, we then sought to investigate the best method of fixation for murine hearts using TEM. Qualitative assessment of myocardium showed that the perfusion of hearts with fixative, after a washout with saline, mostly preserved the cardiac mitochondria from fragmentation or pathologies such as hypercontracture or myocardial disarray (see Figure 3-6 A-C). In addition, quantitative analysis of the data also showed that there was a significant difference between hearts perfused with fixative in terms of cellular pathologies illustrated in

Figure 3-6 D (mean ± S.E group c: 5.67 ± 5.67 vs group a: 94.44 ± 5.56 and group b

94.44 ± 5.56, p<0.0498) and mitochondrial fragmentation shown in Figure 3-6 E.

(group c: 17.00 ± 0.00 versus group a: 94.44 ± 5.56 and group b 83.33 ± 9.62, p<0.0446). Hence immediate perfusion of hearts with fixative can efficiently preserve mitochondrial morphology as well as retaining the structure of cardiomyocytes.

74

Figure 3-6 Different Methods of Fixation and Their Impact on Cardiomyocytes. Representative TEM micrographs of the group a (saline perfusion and direct submersion into the fixative), b (saline perfusion and direct submersion of the dissected ventricular section into the fixative) and c (saline perfusion followed by fixative perfusion), are shown in images A, B and C, respectively. D and E depict the extent of myocardial disarray and mitochondrial fragmentation, respectively.

* and $ denote P<0.05 vs. group a and group b, respectively (N=3 per group).

3.3.3 Subpopulation Specific Differences in Mitochondrial Shape Parameters*

Ultrastructure assessment of cardiomyocytes structure showed minimal alteration in response to 20 minutes of global ischemia. Overall no substantial alterations in terms of sarcomeric length (measured by the Z-line spacing) were present between different study groups (Mean ± S.E ST: 2.37 ± 0.27 Vs. ST+IPC: 2.30 ± 0.29

Vs. ST+ IS: 2.16 ± 0.36 Vs. ST + IPC + IS: 2.14 ± 0.22, ANOVA P=0.4799). We then analysed the three subpopulations of cardiac mitochondria. In mouse cardiomyocytes,

IMF mitochondria reside along the longitudinal axis between the myofibrils and are often in contact with their two neighbouring IMF mitochondria (Figure 3-7 A).

*The results section is descriptive and the exacts values can be found in Table 3.1. 75 Table 3-1 Quantification of Mitochondrial Shape Parameters in Different Subpopulations of Cardiac Mitochondria Using Predicted Mean Values ± S.E. (Modified from (277)).

Mitochondria Log. Perimeter Feret’s Log. Circularity Roundness Subtype Area Diameter AR

IMF 5.78 ± 4020.41 ± 1648.70 ± 0.41 ± 0.64 ± 0.44 ± Vs. 0.32 174.37 75.25 0.02 0.02 0.02 SSM vs. vs. vs. vs. vs. vs. 5.77 ± 3528.59 ± 1350.67 ± 0.26 ± 0.75 ± 0.58 ± 0.33 180.59 *** 77.91*** 0.02*** 0.02*** 0.02***

IMF 5.78 ± 4020.41 ± 1648.70 ± 0.41 ± 0.64 ± 0.44 ± Vs. 0.32 174.37 75.25 0.02 0.02 0.02 PN vs. vs. vs. vs. vs. vs. 5.59 ± 2793.22 ± 1084.1 ± 0.26 ± 0.77 ± 0.58 ± 0.32 *** 177.22 *** 76.47 *** 0.02*** 0.02 *** 0.02 ***

SSM 5.77 ± 3528.59 ± 1350.67± 0.26 ± 0.75 ± 0.58 ± Vs. 0.33 180.59 77.91 0.02 0.02 0.02 PN vs. vs. vs. vs. vs. vs. 5.59 ± 2793.22 ± 1084.1 ± 0.26 ± 0.77 ± 0.58 ± 0.32 *** 177.22 *** 76.47 *** 0.02 0.02 * 0.02

Figure 3-7 Disparate Mitochondrial Subpopulations of Adult Mouse Cardiomyocytes. The stars shown in (A) locate the position of IMF mitochondria whereas in (B) and (C) indicate the position of SSM and PN mitochondria, respectively.

76 Comparatively, SSM and PN mitochondria do not have any specific organisation and are positioned under the cell membrane and around the cell nucleus (Figure 3-7 B and

C), respectively. However, PN mitochondria have a denser population and exhibit higher membrane contact in comparison to the SSM (Figure 3-7 C). In terms of morphological descriptors, IMF mitochondria from ST only treatment have generally larger perimeter and Feret’s diameter in comparison to the SSM and PN mitochondria

(Figure 3-8 B and C and Figure 3-9 E and F), however, their areas resemble those from their SSM counterpart (Figure 3-8 A and Figure 3-9 D). IMF mitochondria are also the least circular and spherical in comparison to

Figure 3-8 Morphometric Analysis of IMF, SSM and PN Mitochondria of Adult Cardiomyocytes. Mitochondrial area, perimeter and Feret’s diameter are illustrated in (A), (B) and (C), while (D), (E) and (F) show mitochondrial roundness, circularity, and aspect ratio, respectively (Modified from (277)).

*** denotes P<0.001 vs. IMF whereas $ and $$$ denote P<0.05 and P<0.001 vs. SSM, respectively (N=6).

77 other two subtypes (Figure 3-8 D, E and F and Figure 3-9 A, B, and C) which may be due to their extensive branching and length. PN mitochondria are generally smaller, shorter, and more circular (Figure 3-8 A, B, C and E and Figure 3-9 C, D, E and F) than the SSM mitochondria but they share the same degree of sphericity (Figure 3-8

D and F and Figure 3-9 A and B). Therefore, the three subtypes of cardiac mitochondria have different shape descriptors and are morphologically unique.

Figure 3-9 Distribution of Mitochondrial Morphometric Descriptors. Frequency distribution of mitochondrial aspect ratio, roundness and circularity are shown in (A), (B), and (C) whereas area, perimeter and Feret’s diameter of IMF mitochondria are shown in (D), (E) and (F), respectively (Modified from (277)).

78 3.3.4 Morphological Responses of IMF Mitochondria*

Having established the morphological differences of the 3 subpopulations of adult cardiac mitochondria, we then studied the responses of individual mitochondrial subpopulations to IPC and ischaemia. IPC itself had a mild effect in terms of altering the integrity of myofibrils and mitochondrial organisation (Figure 3-10 A and B).

However, 20 minutes of ischaemia was enough to trigger mitochondrial fragmentation

(Figure 3-10 C). This effect was not prevented by IPC (Figure 3-10 D).

Figure 3-10 Treatment Based Morphological Responses of IMF Mitochondria. ST and ST followed by IPC are shown in (A) and (B) whereas ST followed by ischaemia with or without IPC are shown in (C) and (D), respectively.

*The results section is descriptive and the exacts values can be found in Table 3.2. 79 Table 3-2 Treatment Based Evaluation of IMF Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277)).

Specific Predicted Mean ± S.E for Different Shape Descriptors Pairwise of IMF Mitochondria after Different Treatments Analysis Log. Perimeter Feret’s Log.AR Circularity Roundness Area Diameter (ST) 5.78 ± 4010.24 ± 1645.08 ± 0.41 ± 0.64 ± 0.44 ± Vs. 0.02 148.94 69.8 0.02 0.02 0.02 (ST + IPC) Vs. Vs. Vs. Vs. Vs. Vs. 5.75 ± 3710.97 ± 1507.18 ± 0.37 ± 0.67 ± 0.47 ± 0.02 147.96 69.4 0.02 0.02 0.02 (ST) 5.78 ± 4010.24 ± 1645.08 ± 0.41 ± 0.64 ± 0.44 ± Vs. 0.02 148.94 69.8 0.02 0.02 0.02 (ST + IS) Vs. Vs. Vs. Vs. Vs. Vs. 5.85 ± 3757.4 ± 1451.33 ± 0.25 ± 0.78 ± 0.60 ± 0.02 148.6 69.66 * 0.02 *** 0.02 *** 0.02 * (ST) 5.78 ± 4010.24 ± 1645.08 ± 0.41 ± 0.64 ± 0.44 ± Vs. 0.02 148.94 69.8 0.02 0.02 0.02 (ST + Vs. Vs. Vs. Vs. Vs. Vs. IPC+IS) 5.83 ± 3647.89 ± 1391.62 ± 0.23 ± 0.78 ± 0.63 ± 0.02 148.41 69.58 * 0.02 *** 0.02 *** 0.02 * (ST + IS) 5.85 ± 3757.4 ± 1451.33 ± 0.25 ± 0.78 ± 0.60 ± Vs. 0.02 148.6 69.66 0.02 0.02 0.02 (ST+ IPC+ Vs. Vs. Vs. Vs. Vs. Vs. IS) 5.83 ± 3647.89 ± 1391.62 ± 0.23 ± 0.8 ± 0.63 ± 0.02 148.41 69.58 0.02 0.02 0.02

Moreover, the assessment of morphological descriptors did not show any significant increase in mitochondrial area and perimeter after different treatments (Figure 3-11 A and B and Figure 3-12 D and E). In addition, IPC itself did not significantly affect the other shape parameters of IMF mitochondria (Figure 3-11 C, D, E and F and Figure 3-

12 A, B, C, F). However, a marked reduction of up to 11% in Feret’s diameter and an increase of 37% in roundness (Figure 3-11 C and D and Figure 3-12 F and B) revealed substantial fragmentation of IMF mitochondria in response to ischaemia. An increase in circularity (Figure 3-11 E and Figure 3-12 C) and a decrease in aspect ratio (Figure

3-11 F and Figure 3-12 A) further supported the extent of ischaemia-induced fission.

Lastly, IPC did not alter the shape changes of IMF mitochondria after ischaemia

(Figure 3-11 C, D, E and F and Figure 3-12 A, B, C, and F). Therefore, the induction of fragmentation of IMF mitochondria after 20 minutes of ischaemia in adult cardiomyocytes was not prevented by the action of IPC.

80

Figure 3-11 Treatment Based Alterations of Shape Parameters in IMF Mitochondria. Measures of size and length including area, perimeter and Feret’s diameter are illustrated in (A), (B) and (C). Roundness, circularity, and aspect ratio are shown in (D), (E) and (F) (Modified from (277)).

* and *** denote P<0.05 and P<0.001 vs. ST, respectively (N=6).

Figure 3-12 Treatment Based Distributions of IMF Mitochondrial Morphometric Descriptors. Frequency distribution of mitochondrial aspect ratio, roundness and circularity are shown in (A), (B), and (C) whereas area, perimeter and Feret’s diameter of IMF mitochondria are shown in (D), (E) and (F), respectively (Modified from (277)).

81 3.3.5 Morphological Alterations of SSM Mitochondria after Ischaemia*

SSM mitochondria have been shown to respond to treatments in a strain- specific manner (186; 276). In this study, IPC alone had no effect on the shape of SSM subtype (Figure 3-13 A and B). Ischaemia-induced fragmentation in this subpopulation of cardiac mitochondria (Figure 3-13 C) and IPC was ineffective in preventing this alteration (Figure 3-13 D).

Figure 3-13 Treatment-Based Morphological Responses of SSM Mitochondria. ST and ST followed by IPC are shown in (A) and (B) whereas ST followed by ischaemia with or without IPC are shown in (C) and (D), respectively.

*The results section is descriptive and the exacts values can be found in Table 3.3. 82 Table 3-3 Treatment Based Evaluation of SSM Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277)).

Specific Predicted Mean ± S.E for Different Shape Descriptors Pairwise of SSM Mitochondria after Different Treatments Analysis Log. Perimeter Feret’s Log.AR Circularity Roundness Area Diameter

(ST) 5.77 ± 3520.92 ± 1348.94 ± 0.26 ± 0.75 ± 0.58 ± Vs. 0.02 100.08 41.37 0.01 0.01 0.02 (ST + IPC) Vs. Vs. Vs. Vs. Vs. Vs. 5.74 ± 3292.36 ± 1254.75 ± 0.25 ± 0.77 ± 0.59 ± 0.02 98.82 40.91 0.01 0.01 0.02

(ST) 5.77 ± 3520.92 ± 1348.94 ± 0.26 ± 0.75 ± 0.58 ± Vs. 0.02 100.08 41.37 0.01 0.01 0.02 (ST + IS) Vs. Vs. Vs. Vs. Vs. Vs. 5.83 ± 3495.68 ± 1295.90 ± 0.19 ± 0.82 ± 0.67 ± 0.02 99.48 41.16 0.01 *** 0.01 *** 0.02 ***

(ST) 5.77 ± 3520.92 ± 1348.94 ± 0.26 ± 0.75 ± 0.58 ± Vs. 0.02 100.08 41.37 0.01 0.01 0.02 (ST + Vs. Vs. Vs. Vs. Vs. Vs. IPC+IS) 5.77 ± 3289.5 ± 1215.07 ± 0.19 ± 0.82 ± 0.67 ± 0.02 98.50 40.78 * 0.01 *** 0.01 *** 0.02 ***

(ST + IS) 5.83 ± 3495.68 ± 1295.90 ± 0.19 ± 0.82 ± 0.67 ± Vs. 0.02 99.48 41.16 0.01 0.01 0.02 (ST+ IPC+ Vs. Vs. Vs. Vs. Vs. Vs. IS) 5.77 ± 3289.45 ± 1215.07 ± 0.19 ± 0.82 ± 0.67 ± 0.02 98.50 40.78 0.01 0.01 0.02

There was an absence of any substantial and meaningful alteration in terms of SSM mitochondrial size and length descriptors after individual treatments (Figure 3-14 A,

B and C and Figure 3-15 D, E, and F). However, reduction of aspect ratio (Figure 3-

14 F and Figure 3-15 A) and increase in circularity (Figure 3-14 E and Figure 3-15 C) and roundness (Figure 3-14 D and Figure 3-15 B) indicated the presence of ischaemia- induced fragmentation. In addition, IPC alone or IPC, given prior to ischaemia, did not influence mitochondrial morphology (Figure 3-14 A-E and Figure 3-15 A-E). Hence, fragmentation caused by ischaemia in SSM mitochondria could not be reversed by the action of IPC.

83

Figure 3-14 Treatment-Based Alterations of Shape Parameters in SSM Mitochondria. Measures of size and length including area, perimeter and Feret’s diameter are illustrated in (A), (B) and (C). Roundness, circularity, and aspect ratio are shown in (D), (E) and (F) (Modified from (277)).

* and *** denote P<0.05 and P<0.001 vs. ST, respectively (N=6).

Figure 3-15 Treatment Based Distributions of SSM Mitochondrial Morphometric Descriptors. Frequency distribution of mitochondrial aspect ratio, roundness and circularity are shown in (A), (B), and (C) whereas area, perimeter and Feret’s diameter of SSM mitochondria are shown in (D), (E) and (F), respectively (Modified from (277)).

84 3.3.6 Distinct Morphological Changes of PN Mitochondria after Ischaemia*

PN mitochondria are the least studied subpopulation of mitochondria in adult heart (165). IPC alone did not induce any morphological changes in PN mitochondria

(Figure 3-16 A and B). In contrast to IMF and SSM mitochondria, ischaemia mediated the formation of circular and swollen mitochondria shown by yellow arrows in Figure

3-16 C, which were also present in the group receiving IPC before ischaemia (Figure

3-16 D).

Figure 3-16 Treatment Based Morphological Responses of PN Mitochondria. ST and ST followed by IPC are shown in (A) and (B) whereas ST followed by ischaemia with or without IPC are shown in (C) and (D), respectively. Yellow arrows indicate the abnormally circular and enlarged mitochondria.

*The results section is descriptive and the exacts values can be found in Table 3.4. 85 Table 3-4 Treatment Based Evaluation of PN Mitochondrial Shape Parameters Using Predicted Mean Values ± S.E. (Modified from (277)).

Specific Predicted Mean ± S.E for Different Shape Descriptors Pairwise of PN Mitochondria after Different Treatments Analysis Log. Perimeter Feret’s Log.AR Circularity Roundness Area Diameter (ST) 5.52± 2780.02 ± 1078.86 ± 0.26 ± 0.77 ± 0.58 ± Vs. 0.03 87.75 35.89 0.01 0.01 0.01 (ST + IPC) Vs. Vs. Vs. Vs. Vs. Vs. 5.62 ± 2783.01 ± 1062.37 ± 0.23 ± 0.8 ± 0.61 ± 0.03 87.27 35.67 0.01 0.01 0.01 (ST) 5.59± 2780.02 ± 1078.86 ± 0.26 ± 0.77 ± 0.58 ± Vs. 0.03 87.75 35.89 0.01 0.01 0.01 (ST + IS) Vs. Vs. Vs. Vs. Vs. Vs. 5.69 ± 2920.72 ± 1084.41 ± 0.18 ± 0.84 ± 0.68± 0.03 * 88.47 36.16 0.01 *** 0.01 *** 0.01 *** (ST) 5.59± 2780.02 ± 1078.86 ± 0.26 ± 0.77 ± 0.58 ± Vs. 0.03 87.75 35.89 0.01 0.01 0.01 (ST + Vs. Vs. Vs. Vs. Vs. Vs. IPC+IS) 5.71 ± 3055.42 ± 1131.47 ± 0.19 ± 0.83± 0.68 ± 0.03 ** 86.95 * 35.58 * 0.01 *** 0.01 *** 0.01 *** (ST + IS) 5.67 ± 2920.72 ± 1084.41 ± 0.18 ± 0.84 ± 0.68 ± Vs. 0 .03 88.47 36.16 0.01 0.01 0.01 (ST+ IPC+ Vs. Vs. Vs. Vs. Vs. Vs. IS) 5.71 ± 3055.42 ± 1131.47 ± 0.19 ± 0.83± 0.68 ± 0.03 86.95 35.58 0.01 0.01 0.01

Consistent with the other two subtypes of cardiac mitochondria, IPC itself did not induce any changes in mitochondrial shape (See Figure 3-17 and 3-18 A-F). However, in support of the TEM observations, there was a significant increase in PN mitochondrial area (Figure 3-17 A and Figure 3-18 D), circularity (Figure 3-17 E and

Figure 3-18 C), and roundness (Figure 3-17 D and Figure 3-18 B) as well as a reduction of aspect ratio (Figure 3-17 F and Figure 3-18 A) in groups receiving ischaemia with or without IPC. Interestingly, the increase in size was followed by an increase in perimeter in the group receiving IPC prior to ischaemia (Figure 3-17 B and Figure 3-

18 E). In addition, there was no alteration in mitochondrial Feret’s diameter after all treatments (Figure 3-17 C and Figure 3-18 F). Overall, the enlargement of PN mitochondria indicates the subpopulation-specific response of cardiac mitochondria to ischaemia.

86

Figure 3-17 Treatment Based Alterations of Shape Parameters in PN Mitochondria. Measures of size and length including area, perimeter and Feret’s diameter are illustrated in (A), (B) and (C). Roundness, circularity, and aspect ratio are shown in (D), (E) and (F) (Modified from (277)).

*, ** and *** denote P<0.05, P<0.01 and P<0.001 vs. ST, respectively (N=6).

Figure 3-18 Treatment Based Distributions of PN Mitochondrial Morphometric Descriptors. Frequency distribution of mitochondrial aspect ratio, roundness and circularity are shown in (A), (B), and (C) whereas area, perimeter and Feret’s diameter of SSM mitochondria are shown in (D), (E) and (F), respectively (Modified from (277)).

87 3.3.7 Morphological Shape Alterations of IMF Mitochondria in Mfn1/2 DKO

As discussed in section 1.2, alteration in the expression of mitochondrial fusion and fission proteins in the adult heart can influence mitochondrial shape. Genetic ablation of both mitofusins was followed by a drastic shortening of mitochondrial length and a change in their organisation (Figure 3-19 c and d) in comparison to the

WT mitochondria (Figure 3-19 a and b). Mitofusin deficiency was also accompanied by the formation of abnormally asymmetric and dense cristae thereby indicating the mitofusin deficiency affect both the shape and structure of cardiac mitochondria.

Figure 3-19 Alteration of Mitochondrial Morphology in MFN1/2 DKO Mice. (a) and (b) show the mitochondrial organisation in a WT cardiomyocyte whereas (c) and (d) demonstrate mitochondria from a MFN1/2 DKO cardiomyocyte. (a) and (c) were taken at 10k whereas (b) and (d) were captured at x50k magnification.

88 We further evaluated the effect of mitofusin ablation by quantifying the shape alterations in IMF mitochondria. Mitofusin deficiency caused a drastic decrease in mitochondrial size in terms of area (Figure 3-20 a and Figure 3-21 a, predicted mean

± S.E WT: -0.88 ± 0.06 vs. -1.12 ± 0.06, p<0.003) as well as perimeter (Figure 3-20 b and Figure 3-21 b, WT: 1.10 ± 0.03 vs. 0.94 ± 0.03, p<0.0001) and induced mitochondrial shortening (Figure 3-20 c and Figure 3-21 c, WT: 0.15 ± 0.03 vs. -0.01

± 0.03, p<0.0001). Mitochondrial branching was also reduced due to the significant

Figure 3-20 Mitochondrial Shape Descriptors in WT and Mfn1/2 DKO Mice. (a), (b) and (c) show the mitochondria area, Perimeter and Feret’s diameter whereas (d), (e) and (f) illustrate the mitochondrial roundness, aspect ratio and circularity of WT and Mfn1/2 DKO mitochondria, respectively.

** and *** denote P<0.01 and P<0.001 vs. WT, respectively (N=3).

89 increase in mitochondrial circularity (Figure 3-20 f and Figure 3-21 e, WT: 0.60 ± 0.03 vs. 0.65 ± 0.03, p<0.004). However, sphericity related measures including roundness

(Figure 3-20 d and Figure 3-21 d, WT: 0.49 ± 0.02 vs. 0.51 ± 0.02, p<0.361) and aspect ratio (Figure 3-20 e and Figure 3-21 f, WT: 0.81 ± 0.04 vs. 0.75 ± 0.04, p<0.313) were unchanged. Overall, these data indicated that the ablation of mitofusin proteins, which govern mitochondrial fusion, can alter the mitochondrial morphology, and further emphasise the importance of mitochondrial shape descriptors to comprehensively quantitate these alterations.

Figure 3-21 Distributions of IMF Mitochondrial Morphometric Descriptors in WT and Mfn1/2 DKO Mice. Logarithmically transformed area, perimeter, Feret’s diameter and aspect ratio are shown in (a), (b), (c) and (f). Roundness and circularity of PN mitochondria are shown in Figure (d) and (e).

90 Discussion

In this chapter, we primarily looked at different fixation techniques and concluded that the best method to preserve murine heart ultrastructure is the immediate fixative perfusion after the initial saline wash. Having established the fixation methodology, we then went further and evaluated the morphological heterogeneity of three subsets of adult cardiac mitochondria and documented that they are morphologically different. We also showed that the mitochondrial shape descriptors are important to the adult cardiomyocytes since they can be affected by pathological conditions such as ischaemia and the absence of mitochondrial dynamic proteins, mitofusins. Finally, we showed that the changes of morphological shape descriptors after ischaemia cannot be reversed by IPC.

The preparation of tissue for fixation is one of the fundamental steps of 2D electron microscopy. The immersion of tissue in a fixative without perfusion has been suggested to delay the process of fixation and hence alter the tissue structure (278).

Accordingly, we found that immersion of a section of cardiac tissue or the whole heart into the fixative, without prior perfusion, induced severe deformities in the cell integrity and mitochondrial morphology. In contrast and consistent with a previous report we also found that the perfusion of the heart with the fixative prevents any ultrastructural alteration at the cellular level (279). Therefore, perfusion-based fixation should be used to allow perfect preservation of mitochondrial structure for 2D electron microscopy assessment.

The notion that the cardiac mitochondria exist in three unique subpopulations was challenged recently (195). Rassaf et al. (2017) made a qualitative evaluation of mitochondrial shape in 2D and concluded that cardiac mitochondria do not separate

91 into different subpopulation (195). This can be partially due to the limited methodologies for the evaluation of mitochondrial shape. In contrast to Rassaf paper and consistent with a previous report in mouse skeletal muscle we found numerous differences between mitochondrial subpopulation (170). IMF mitochondria are longer and less spherical than the other subtypes of mitochondria (170). However, a study on monkey myocardium revealed the larger size of SSM mitochondria than those from

IMF suggesting that although morphological characteristics of mitochondrial subpopulation may be preserved in different tissues, it varies across species (171).

Some of the shape parameters of individual mitochondrial subpopulation vary across different animal strains and different tissues. For instance, the assessment of mitochondrial shape at normal status revealed that mouse IMF mitochondria have a

Feret’s diameter of approximately 1.6µm which is in the same range to those from

Japanese monkeys but shorter than the IMF mitochondria from feline ventricular tissue

(168; 170). In addition, mitochondria from mouse skeletal muscle are shorter than those from the mouse heart which can be due to the higher ATP demand in the heart thereby signifying the potential adoption of mitochondrial shape to function (280).

The morphological heterogeneity of cardiac mitochondria is also evident in terms of their response to ischaemia. Specifically, we observed a unique increase in the mitochondrial area of PN mitochondria. Consistent with this finding the area of PN mitochondria from rat heart has been shown to increase when subjected to hypoxic stress (176). Moreover, an early qualitative study by Jennings et al. (1974) showed that both IMF and SSM mitochondria from dog’s heart undergo extensive enlargement after 40 minutes of ischaemia (281). However, later studies indicated that IMF mitochondria from Sprague Dawley rats undergo less swelling after ischaemia in comparison to the SSM (187). In contrast, and consistent with our findings no effect

92 in both subtypes were seen in terms of mitochondrial size after ischaemia in rabbit heart (186). Taken together these data suggest a species-specific response of mitochondrial subpopulation to ischaemia and open new avenues for investigations into the morphological response of human mitochondria to ischaemia.

IPC treatment has been shown to maintain mitochondrial function during ischaemia but whether this mechanism is achieved by the regulation of mitochondrial dynamics is inconclusive (187). Unlike previous reports (252; 254), we did not observe any preservation of mitochondrial morphology in mouse cardiomyocytes treated with

IPC prior to ischaemia. Conversely and despite not seeing any significant effect on the mitochondrial shape or Drp1 expression, a study by Cellier et al. (2015) concluded that remote IPC preserves mitochondrial dynamics. Similarly, the work by Pride et al.

(2014) showed that pharmacological preconditioning using nitrate increases Drp1 phosphorylation which then protects against IRI injury in the heart (254). However, the authors did not document whether this reduction was via preservation of mitochondrial shape or fission/fusion events in the actual rat hearts. Having mentioned that, our work was limited only to ischaemia and we did not investigate the possible protective effects of IPC after reperfusion. Hence, this fact may partially explain the differences between our data and previous IPC-based studies incorporating the effect of mitochondrial dynamics in IRI.

To further investigate the mitochondrial shape changes in adult cardiomyocytes we used mice deficient in mitofusins. In line with a previous report, we found a significant reduction in the mitochondrial area of Mfn1/2 DKO mice (198).

In addition, consistent with our findings, Mfn1/2 DKO mice had been reported to have increased mitochondrial volume density, abnormal cristae and rounded/more spherical mitochondria (200), which were all associated with dysfunction of cardiac

93 mitochondria. Overall, these data prove that the changes in mitochondrial morphology are important to the adult heart and can be used to distinguish between normal and pathological conditions.

Conclusion

In summary, we have found that the three subpopulations of cardiac mitochondria have different shape descriptors and respond uniquely to pathological conditions such as ischaemia and the absence of mitofusin proteins. Although IPC has been suggested to inhibit mitochondrial fission prior to IRI, it did not preserve the mitochondrial shape after ischaemia.

94 Chapter 4. Quantification of Mitochondrial Dynamics in 3D: Shape Descriptors of Mitochondrial Morphology in 3D

Introduction

Mitochondria are the main cellular sources of energy production and play a pivotal role in cardiac homeostasis (17). Mitochondria meet cellular demands by altering their morphology and network organisation, which in turn modulates their metabolism (282). Cardiac mitochondria are tubular organelles that extend throughout the cell and make up 30% of its volume (283). Their network plasticity is governed by the action of fission and fusion proteins since the deficiency in these proteins severely impact their structure (156; 198; 199; 204; 243; 250). Despite being non-flat organelles and possessing a 3D shape, the morphological examination of mitochondrial network integrity, as well as single mitochondrion characteristics, are often performed in 2D

(171; 197). The main issue with the 2D approach is that it can omit the 3D orientation of the organelle. Also, the way the specimen is sliced can be different at individual 3D slices that cover the entire length of the mitochondria (See Figure 4-1). Likewise, the mitochondrial shape is sometimes measured based on the position of the Z-line and the sarcomeric length which can also be affected by the 2D analysis (7). Despite these limitations, techniques to assess mitochondrial shape in 3D can be also relatively crude, since they are mainly based on diffraction-limited light microscopy, and sometimes require the in vitro assessment of mitochondria which involves their extraction from cardiomyocytes (201; 271; 284). Hence, electron microscopy-based

3D analysis can currently provide the most relevant measure of mitochondrial shape in adult cardiomyocytes.

The absence of mitofusins has been shown to affect mitochondrial morphology in cardiomyocytes but the exact effects of mitofusin ablation on mitochondrial 3D

95 shape is unknown. In addition, cardiac contractility in terms of Ca2+ handling and mitochondrial respiration relies heavily on the interaction between junctional SR (j-

SR) and mitochondria. This interplay is regulated by the tethering action of Mfn2 and may contribute to the mitochondrial 3D shape (39; 199).

Figure 4-1 2D and 3D Shape of an Adult Cardiac Mitochondrion. All slices from the top, middle and bottom right to left depict the alteration of the 2D shape of a mitochondrion based on its orientation and cutting angle. The last slice at the bottom right illustrates the 3D shape of the mitochondrion. Scale bar is 2000 nm.

96 Mitochondria from Mfn2 KO mice are enlarged and exhibit altered Ca2+ uptake and release when subjected to caffeine stimulation, which is mainly due to the dissociated j-SR mitochondria interaction (199). Despite the presence of physiological evidence of abrupt calcium signalling in Mfn2KO mice, there has not been any anatomical explanation for this malfunction (199; 201). This may be due to the assessment of mitochondrial-jSR interplay in 2D which in fact occurs in three dimensions and hence has brought up controversies in terms of the exact tethering role of Mfn2 (199; 201;

245; 285).

As mentioned earlier in the introduction, there are several techniques that are used to study the 3D nature of different organelles inside cardiomyocytes. STS-TEM is performed using the manual sectioning of the processed tissue and can achieve sub- nanometre resolution. Sections are then collected and stained before imaging. One of the main limitations of this method is the collection of the tissue which may results in the slice puncture, overstretching or even loss of the sectioned specimen (286).

Volumetric assessment of tissue using STS-TEM can be time-consuming and labour intensive due to the relative sectioning and image registration required prior to reconstruction (287). To overcome these challenges several automated techniques such as SBF- or FIB-SEM have been developed. The SBF-SEM involves the preparation of tissue in high conductivity resin and its attachment to a surface of a pin.

The specimen is then held still inside a SEM chamber and cut using a diamond knife.

Images that are formed by backscattering of the heavy metals inside the processed tissue are then captured after each cut (289). Comparatively, FIB-SEM uses ion beams to mill the surface of the specimen and the images are then taken by backscattering

(290). These two automated methods allow the acquisition of larger volumes and better slice registration for 3D reconstruction, however, they have their own disadvantages

97 (288). Unlike STS-TEM, SBF- and FIB-SEM are very expensive and the tissue slices are destroyed after imaging. Both techniques are also limited in terms of field of view

(196). Tissue specimens that are prepared for SBF-SEM can be frequently used, however, the build of charge on the surface of the block and warping artefact can limit capturing larger 3D volumes as well as limiting the axial resolution to the maximum value of 25 nm (288; 291). Conversely, FIB-SEM does not have the mentioned drawbacks of SBF-SEM and can be used to obtain 3D stacks with a resolution of up to 4nm in voxel size; however, this method is destructive and the sample can be only used once (288; 292). Until recently, the assessment of cardiac-related 3D samples was mainly performed using manual sectioning which as mentioned is far too time- consuming (286). These major challenges have been recently overcome by using FIB- and SBF-SEM which have allowed more detailed visualisation of organelle compartmentalisation in cardiomyocytes (261; 262). While studies incorporating these techniques have paid much attention to the component of Ca2+ signalling including SR and t-tubules, the relevance of 3D organisation and morphology of mitochondria to the adult cardiomyocytes has not been thoroughly explored.

Here we initially investigated the relevance of mitochondrial 3D shape and mitochondrial 3D interaction with SR in Mfn2 KO and WT mice using STS-TEM. We then further explored the benefits of using automated techniques including FIB- and

SBF-SEM to obtain 3D stacks and study the structural and morphological characteristics of adult cardiac mitochondria.

Methods

4.2.1 Materials/Mouse Strains

All animal experiments were performed in compliance with the Animals

98 (Scientific Procedures) Act 1986 published by the UK Home Office. Male 8-10 weeks

C57/BL6 mice were used for general assessment of mitochondrial structure and organisation using FIB-SEM and SBF-SEM. Female 8-10 weeks Mfn2 loxp/loxp mice crossed onto specific Myh6 nuclear-directed ‘turbo’ Cre (30; 199; 293) and WT littermates were used for the STS-TEM and were obtained by kind collaboration with

Prof Gerald Dorn from Washington University.

4.2.2 Genotyping

For genotyping, ear snips from each animal were lysed (DirectPCR Lysis

Reagent (402-E/Viagen)) and mixed with primers recognising the Cre-gene and content of Taq DNA Polymerase (Qiagen-201203) kit. Subsequently, samples underwent electrophoresis and were imaged using Odyssey imaging system for the detection of Cre gene as shown in Figure 4-2.

Figure 4-2 Example of Genotyped Samples. The Mfn2 KO mice express the Cre gene (white bands in the centre of the image) and WT littermates do not possess the Cre gene. (modified from (294)).

99 4.2.3 SBF- and FIB-SEM Imaging

For SBF-SEM, ventricular tissues from a male C57/BL6 mouse were processed according to the Mark Ellisman Protocol for SBF-SEM (295). Processed tissues were mounted on an aluminium pin and trimmed to 1.0 mm x 1.0 mm before being silver painted. The Specimen were then placed in a Gatan 3 view sample holder within a

Zeiss Sigma variable pressure field emission SEM and cut using an automated diamond knife with the thickness of 100nm. Images were taken at a magnification resulting in 32 x 32 nm pixel size in the X and Y axis. This work was performed in collaboration with Dr Peter Munro from the UCL Institute of Ophthalmology.

For FIB-SEM, a mouse heart was prepared based on methods described in section 3.2.3 and the sample was cut, imaged, and aligned using Zeiss FIB-SEM at a resolution of 18.6 x 18.6 x 60 nm in X, Y, and Z axis, respectively. This work was performed in collaboration with Rebecca W. Poh from Carl Zeiss Pte. Ltd, Singapore.

4.2.4 Sample Preparation and Imaging for STS-TEM

Mfn2 and WT hearts (N=5) were processed as described in section 3.2.3. For

3D evaluations, 40 consecutive slices from 3 different regions that included IMF mitochondria were sectioned (with the thickness of 70nm) for each heart and stained with lead citrate. Mitochondria within these 40 slices were reconstructed and imaged

(2.86 nm X 2.86nm pixel size in X and Y) using Tecnai G2 Spirit TEM from FEI, equipped with an Olympus-SIS Morada CCD camera. This section was performed in collaboration with Mr Ian White from the MRC Laboratory of Molecular Cell Biology,

UCL.

100 4.2.5 3D Image Processing and Data Acquisition

3D reconstructions, alignment (for STS-TEM) and shape analysis were performed using the Amira Software. For STS-TEM, mitochondria present in their entirety within the 40 slices were segmented and reconstructed as shown in Figure 4-

3 A and B. In total 219 Mfn2 KO mitochondria were reconstructed and compared to their 317 WT counterpart. For FIB-SEM, 50 randomly selected mitochondria from a cardiomyocyte, covering 3 different mitochondrial subpopulations, were reconstructed, and analysed.

Shape quantification was performed via multi component module of Amira software and the following parameters were used for evaluation of mitochondrial morphology:

a. Anisotropy (Roundness) - ‘1 minus the ratio of the smallest to the largest

eigenvalue of the covariance matrix. This parameter measures a region's

deviation from a rounded shape’

b. Elongation - ‘the ratio of the medium and the largest eigenvalue of the

covariance matrix’ with values close to zero representing more elongated

objects;

c. Flatness - ‘the ratio of the smallest and the medium eigenvalue of the

covariance matrix. Flat objects have small values close to ‘0’.

d. Volume - ‘the volume of the region in units of the voxel size’;

Mitochondria and jSR minimum distance was measured using an Image J (V. 1.46r)

‘ucl_2LineDist’ plug-in, which was a kind gift from Mr Daniel Ciantar. Mitochondria and jSR networks were traced in each 40 sections series. For each

101

Figure 4-3 A Representative Reconstruction of Mitochondria, T-tubules and the

j-SR. j-SR (Green), T-tubules (pink) and mitochondria (Yellow, blue and red colour) of a WT mouse shown in (B) were made based on the original TEM micrographs (A) (modified from (294)). Scale bars are 500nm. network, two lines were hand-drawn between jSR and mitochondria in 2D and the plugin randomly drew 20 perpendicular lines (See Figure 4-4 A and B) between the two organelles to calculate the minimum distance (gaps exceeding 50nm range were excluded (199)).

102

Figure 4-4 Mitochondria-jSR Distance Measurement. (A) shows a non-segmented diagram whereas (B) depicts the measured distance. The white arrow indicates the position of the two lines on the surface of mitochondria and j-SR as well as lines drawn between them by ‘ucl_2LineDist’ plug-in (modified from (294)). 4.2.6 Statistics

Statistical tests were performed using Stata (v.13). To address heart and cell variability the hierarchical nested mixed effects multi-level regression was used to assess the disparities between 3D morphological descriptors of WT and Mfn2 KO mice. The same test was also used to assess mitochondria - jSR interaction. The statistical modelling was performed in collaboration with Dr Qiao Fan, Dr Bibhas

Chakraborty (from Centre for Quantitative Medicine, DUKE-NUS) and Miss Jackie

Cooper (from Institute of Cardiovascular Science, UCL). Results of the nested model were reported as predicted means and 95% confidence intervals and graphs presenting these results were made using Microsoft Excel 2010. Differences between shape parameters of different mitochondrial subpopulation were analysed using one-way

ANOVA. P-value of ≤0.05 was considered significant.

103 Results

4.3.1 Alteration of Mitochondrial 3D Shape Descriptors in Mfn2 KO

Mitochondria

The absence of Mfn2 has been shown to dramatically alter mitochondrial size

(199). The evaluation of mitochondria from Mfn2 KO mice using the STS-TEM revealed that they had a larger volume (Predicted mean ± S.E.: 0.87 ± 0.08 µm3 Mfn2

KO vs. 0.61 ± 0.07 µm3 WT, p<0.001, Figure 4-5 A and Figure 4-6 D).

Figure 4-5 Influence of Mfn2 Ablation on Mitochondrial Shape Descriptors. (A) and (B) indicate mean and confidence interval of mitochondrial volume and elongation whereas (C) and (D) depict mitochondrial roundness and flatness, respectively (modified from (294)).

* and *** denote P<0.05 and P<0.001 vs. WT, respectively (N=5).

104 Mfn2 KO mitochondria were also flatter (0.51 ± 0.03 Mfn2 KO vs. 0.46±0.03 WT, p<0.001, Figure 4-5 D and Figure 4-6 B) but almost similarly elongated (0.40±0.02

Mfn2 KO vs. 0.36±0.02 WT, p<0.080; Figure 4-5 B and Figure 4-6 C). In addition,

Mfn2 KO mitochondria exhibited a lower level of roundness (0.80±0.02 Mfn2 KO vs.

0.83±0.02 WT, p<0.021; Figure 4-5 C and Figure 4-6 A) in comparison to WT mice.

Taken together these results indicate the importance of mitochondrial 3D shape descriptors to predict morphological changes that were developed by Mfn2 deficiency.

Figure 4-6 Distributions of Mitochondrial 3D Shape Descriptors in WT and Mfn2 KO Mice. (A) and (B) illustrate the distributions of mitochondrial roundness and flatness whereas (C) and (D) show the distribution of mitochondrial elongation and volume, respectively (modified from (287)).

105 4.3.2 Association of Mitochondrial 3D Shape Descriptors

The correlative interdependence assessment of mitochondrial shape parameters can indicate the importance of individual mitochondrial parameter to uniquely describe the 3D shape. Here we observed no association between volume versus flatness (Rho (p-value): 0.0007 (0.986)), roundness (-0.0359 (0.406)), or elongation

(0.0551 (0.202)). Similarly, elongation and flatness did not show any degree of association (Rho (p-value): -0.0260(0.547). However, there was a negative correlation between roundness versus elongation (-0.795 (0.0001)) and vs. flatness (-0.5142

(0.0001). Thus, the absence of any correlation between mitochondrial volume and other parameters demonstrate the importance of shape parameters to independently and uniquely describe mitochondrial morphology.

4.3.3 Anatomical Distribution of Mitochondria-jSR Network in Mfn2 KO Mice

Mfn2 ablation has been shown to induce ER stress and alter cardiomyocyte

Ca2+ signalling (199; 208). Mfn2 is known to act as a tether linking ER to mitochondria, however, whether the absence of Mfn2 increases or decreases mitochondria/ER association is controversial (245; 285). In line with the previous functional studies, we found a significant increase in the mitochondria - jSR distance in Mfn2 KO mice (Predicted mean ± S.E.: WT: 12.57 ± 0.24 nm vs. 16.75 ± 0.29 nm

Mfn2, p<0.0001; Figure 4-7 A). In addition, there was a substantial reduction in the number of mitochondria – jSR networks number in Mfn2 KO mice versus WT (WT:

18.45 ± 0.94 vs. 9.30 ±1.06 Mfn2 KO, p=0.0001; Figure 4-7 C). Although we did not investigate the length of the interface between the mitochondria and SR in 2D, the depth of interface along Z-axis was unchanged (WT: 226.24 nm ± 14.29 vs. 207.10 nm ± 15.63 Mfn2 KO, p<0.064; Figure 4-7 B). Therefore, Mfn2 ablation severely

106 alters the number of mitochondria and jSR network and decreases anatomical association between the two organelles.

Figure 4-7 Influence of Mfn2 Ablation on Mitochondria-jSR Network Characteristics (from STS-TEM data). Mitochondria j-SR distance measurements are shown in (A) while the length of mitochondria-jSR network interface along the z-axis, as well as their number in different genotypes, are shown in (B) and (C), respectively (modified from (294)).

*** denotes P<0.001 vs. WT (N=5 per group).

4.3.4 Evaluation of Mitochondrial Morphology and Network Structure Using

SBF-SEM

Having quantified mitochondrial morphology using STS-TEM, we then sought to investigate the use of SBF-SEM for visualising the mitochondrial network organisation in the entire cell. Using SBF-SEM we could visualise and reconstruct the

107 whole network of cells with their structure. Remarkably, a considerable gap at µm range, shown in white in Figure 4-8 A, was evident between the cardiomyocytes.

Upon further segmentation, we realised that this lumen was mostly filled with capillaries (See Figure 4-8 B).

Figure 4-8 Reconstruction of a Cardiac Myocytes. (A) Shows cardiac cells structure and their defined striations within the heart tissue. (B) illustrates the reconstruction of the capillaries, shown in red, surrounding the cells with grey striations. Scale bars are 9µm. Cardiac mitochondria have been suggested to be separate entities that are restricted and surrounded by myofibrils (296; 297). Unlike this fact, reconstruction of the mitochondrial network within the cardiomyocyte revealed the extensive interconnectivity of cardiac mitochondria that formed large tubule-like networks shown in Figure 4-8 A. These tubules were arranged longitudinally in rows contacting

108 the nucleus as well as the cell membrane and filled around 50% of the cell volume.

Interestingly, mitochondria were less populated at the poles of the cell and were mostly fragmented in these regions Figure 4-9 B.

Figure 4-9 Mitochondria Network in Cardiac Myocytes. (A) depicts the mitochondrial network morphology in light blue colour. This image also illustrates the position of mitochondria to the cell membrane (light gold) and cell nucleus (green). (B) illustrates the fragmented mitochondria at the pole of the reconstructed cardiac myocyte. Scale bars are 9µm. Furthermore, network analysis revealed that the mitochondria present in rows were sometimes encircled by the mitophagosomes that often are associated with dark colour due to their high lipid content (See Figure 4-10 A). Mitophagosomes formed helices and were often extended to cover the entire length of the mitochondrion (See

Figure 4-10 B). These autophagosomes were not limited to individual mitochondria and were sometimes present in the neighbouring mitochondria (Figure 4-9 C). This

109 fact suggests that the process of autophagy is not limited to single mitochondria and can affect the entire mitochondrial network. Taken together, using the SBF-SEM we showed that mitochondria form an interconnected network throughout the cardiomyocytes and the integrity of this network is regulated by the quality control function of autophagosomes.

Figure 4-10 Autophagosome Formations along the Mitochondrial Network. (A) shows the encirculation of mitochondria by mitophagosome that exhibit the distinct darker colour. (B) Depicts the helical structure of a reconstructed mitophagosomes (yellow) whereas (C) shows their interaction with the surrounding network of mitochondria (shown in red). Scale bar in (A) is 4000 nm and they are 9000 nm in (B) and (C).

110 4.3.5 Evaluation of Mitochondrial Morphology and Network Structure Using

FIB-SEM

One of the drawbacks of SBF-SEM in comparison to the FIB-SEM is its limitation in terms of slice thickness (286). Due to the build-up of charge on the sample’s surface, we were unable to decrease the Z-resolution below 100 nm in SBF-SEM sample, whereas we easily achieved a Z-resolution of 60 nm with FIB-SEM. Hence, we sought to reconstruct and quantify the 3D shape parameters of different mitochondrial subpopulations of a cardiomyocyte using FIB-SEM. 3D reconstruction of IMF

Figure 4-11 3D Reconstruction of IMF from an adult cardiomyocyte using FIB- SEM. A stack of IMF mitochondria is shown in red. Scale bar is 2000nm.

111 mitochondria revealed that they have adapted to the striated structure of the cardiomyocyte and form cube-like entities packed neatly on top of each other (see

Figure 4-11). Comparatively, SSM mitochondria mainly possess dysmorphic network organisation which helps them fill up space beneath the sarcolemma (See Figure 4-

12).

Figure 4-12 3D Reconstruction of SSM from an adult cardiomyocyte using FIB- SEM. A stack of SSM mitochondria is shown in blue. Scale bar is 1000nm.

PN mitochondria exhibited a globular morphology and formed a grape-like network around the cell nucleus (See Figure 4-13). Taken together, 3D reconstruction of an

112 adult cardiomyocyte using FIB-SEM showed that cardiac mitochondrial subpopulations exhibited disparate 3D network morphology and organisation.

Figure 4-13 FIB-SEM 3D Reconstruction of PN Mitochondria and Nucleus from an Adult Cardiomyocyte. This diagram shows the globular-shaped PN mitochondria (shown in green colour) surrounding the cell nucleus. Scale bar is 1000nm.

Consistent with the mentioned observations, preliminary 3D morphometric assessment of 50 randomly reconstructed mitochondrial subtypes revealed the presence of differences between them. PN mitochondria were smaller in comparison to the other two subtypes, whereas no significant difference was observed between

IMF and SSM (Mean ± S.E.: IMF: 0.87± 0.09, SSM: 1.02± 0.13, PN: 0.46 ± 0.03, p<0.0002, Figure 4-14 A). There were also non-significant differences between

113

Figure 4-14 3D Shape Descriptors of Different Subpopulation of Cardiac Mitochondria. (A) and (B) indicate mean and confidence interval of mitochondrial volume and elongation whereas (C) and (D) depict mitochondrial flatness and roundness, respectively.

** and $$$ denote P<0.01 and P<0.001 vs. IMF and SSM, respectively (N=50 mitochondria per group). mitochondrial subtypes in terms of elongation (IMF: 0.33 ± 0.03, SSM: 0.32 ± 0.03,

PN:0.40 ± 0.2, P<0.1379, Figure 4-14 B), flatness (IMF: 0.43 ± 0.03, SSM: 0.44 ±

0.03, PN:0.48 ± 0.03, P<0.5067, Figure 4-14 C), and roundness (IMF: 0.86 ± 0.01,

SSM: 0.87 ± 0.01, PN:0.84 ± 0.01, P<0.1964, Figure 4-14 D). These data further revealed that the morphological differences of cardiac mitochondria are not limited to

2D but are also present in 3D.

114 Discussion

In this chapter, we initially showed using STS-TEM that the 3D morphological descriptors of cardiac mitochondria are influenced by Mfn2 deficiency. We additionally presented that the mitochondrial - jSR anatomical interplay is interrupted by the absence of Mfn2 protein. We finally documented that automated techniques such as SBF- and FIB-SEM can be used to study mitochondrial network integrity and morphology inside the adult cardiomyocytes.

Mitochondrial imaging is often performed in 2D (298) Mitochondria are non- flat organelles and their 2D shape varies according to the plane of axis they are imaged

(197; 299)(300). Hence, conclusions of the 3D shape from 2D evaluations may not accurately predict the shape of mitochondria. This can result in the loss of useful data and reduce the sensitivity of the analysis (298). Having mentioned that, 2D image analysis should not be regarded as incomplete and 3D image analysis can be performed alongside the 2D analysis to complement its sensitivity.

When performing 2D or 3D shape analysis, several different shape parameters should be used to increase the sensitivity of the data (301) Here we observed no correlation between the mitochondrial volume and other parameters of shape which indicates that the interpretation of results could have been biased if the results were solely based on mitochondrial volume. In line with this fact, a previous report documented that isolated Mfn2-deficient cardiac mitochondria, which were assessed using FACS, were not different from their WT counterparts in terms of volume. The same authors also did not provide any other measures of shape thereby failing to provide any valid morphological explanation for the observed functional differences

(201). Hence, the 3D evaluation of mitochondrial shape should include a multi-

115 parametric approach since restricting image analysis to one single parameter may lead to a wrong interpretation of study results.

Genetically modified mouse models of proteins involved in mitochondrial dynamics provide a great opportunity to study the changes of mitochondrial shape (30;

200; 203; 205; 212). Unlike other fusion components and consistent with previous reports, the ablation of Mfn2 induced an enlargement of mitochondrial size (199; 207;

212). This enlargement has been shown to contribute to increased Ca2+ retention capacity and subsequent lower MPTP susceptibility in Mfn2 KO IMF mitochondria

(201). Interestingly, the ablation of both mitofusins induces substantial reduction of mitochondrial 2D and 3D size which is suggested to be due to the dominant influence of Mfn1 function on the mitochondrial shape (198; 244). Whether the observed mitochondrial enlargement is due to abrogated ER function to facilitate fission (39;

245) or is due to DNA damage induced by interrupted mitophagic culling (119; 302) in cardiac Mfn2 KO mitochondria is unknown. Moreover and in contrast to previous reports in skeletal muscle and neurons, we observed mitochondria to be less rounded in Mfn2 KO cells (23; 293). Hence, tissue-specific characteristics of mitochondrial morphology, which were described in chapter 3, are applicable to both 2D and 3D parameters. Overall, these facts further demonstrate that the mitochondrial shape evaluation in 3D is as important as the 2D analysis and allows the identification of pathological conditions.

The relationship between mitochondria and ER is fundamental to the regulation of mitochondrial metabolism and its dynamics (39; 303). This relationship is tightly regulated by the function of Mfn2 in the adult heart and cardiac-specific Mfn2

KO mouse models exhibit altered bioenergetics and Ca+2 uptake characteristics (199).

Despite the presence of functional data, there has been no evidence to provide any

116 meaningful anatomical explanation for the observed physiological differences (199;

201). Our work is the first its kind to address this issue in the heart. Previous studies in Mfn2 KO cell lines have shown an increase in ER-mitochondrial distance (245;

304). However, two other reports have claimed that mitochondria-ER distance is increased in the absence of Mfn2 (285; 305). These conflicting results may be due to the assessment of mitochondrial-ER distance in 2D whereas their interplay occurs in

3D, and hence our results may address the reason behind the presence of these inconsistencies.

In this section, we used SBF-SEM and showed some new features of mitochondrial network plasticity in cardiomyocytes. Mitochondria have been postulated to exhibit a disconnected and semi-fragmented network in cardiac cells

(296; 297). In contrast to this notion, we saw an interconnected network of mitochondria in the centre of cardiac cells that were extended towards both poles of the cell. In addition, we observed extended helical mitophagosomes surrounding the mitochondrial network. These helices have been suggested to play a role in the mitochondrial quality control and mitochondrial network alignment in the cardiomyocytes (306). However, the exact impact of mitophagosomes on the shape of adult cardiac mitochondrial is poorly understood. Besides, fragmented mitochondria were mostly present in the poles of the cell. It is possible to speculate that different regional bioenergetics mediate the formation or migration of mitochondria by upregulation mitophagy and fission.

The subcellular location of cardiac mitochondria affects the overall mitochondrial network structure as well as their general morphology (165). Here, using 3D FIB-SEM we demonstrated that the three subpopulations of cardiac mitochondria have distinct 3D morphology. Our results are in partial agreement with

117 the recent FIB-SEM study where they showed the PN mitochondria to have smaller volume in comparison to the other mitochondrial population (307). These results once more indicate a conflict with the study of Rassaf et al. (2017) who claimed that the cardiac mitochondria are homogenous throughout the cell (195). The main problem with the latter study is that unlike their conclusion, there was a substantial lack of any robust quantitative approach to evaluate the mitochondrial subtypes in both 2D and

3D. Lastly, we did not study the 3D morphology of individual mitochondrial subpopulations in the entire cell and automated techniques may help this aspect of 3D quantification.

Conclusion

In conclusion, 3D shape descriptors are the key features of cardiac mitochondria. These parameters can be affected under pathological conditions such as the absence of Mfn2 which interrupts the mitochondria-jSR anatomical interplay and substantially affects the 3D morphology of cardiac mitochondrial morphology. The use of novel automated 3D volumetric electron microscopy including SBF- and FIB-

SIM can overcome the limitations associated with manual 3D methods including sectioning, registration, and the analysed depth. Therefore, automated techniques can greatly enhance our understanding of mitochondrial network organisation and help us explore the morphological dynamics of cardiac mitochondria.

118 Chapter 5. High-throughput Screening of Small Molecule Modulators of Mitochondrial Dynamics

Introduction

Cellular metabolism and survival are dependent on the optimum balance between mitochondrial fission and fusion, and this equilibrium is altered under pathological conditions (7; 19; 22; 88; 282; 308). The pathological shift in this equilibrium is mainly associated with an increase in the occurrence of mitochondrial fission and is linked to numerous diseases including cardiovascular IRI, neurological diseases, acute kidney injury and multi-drug resistant tumours (7; 19; 309–311). As a result, therapeutic targeting of mitochondrial fission has attracted recent interest (7;

312; 313). This notion has also led to an increase in the use and development of high throughput screening (HTS) methods to better understand the protein regulators of mitochondrial dynamics and to discover novel chemical modulators of mitochondrial fission and fusion (135; 273; 314). For instance, using an algorithm capable of assessing mitochondrial shape descriptors, Screaton and his team identified a protein known as Romo1 that is required for oligomerisation of OPA1 protein in primary-cell line (135). In addition, the mitochondrial fission proteins, Mff and MiD49/51, were only discovered recently by screening a Drosophila siRNA collection and screening of uncharacterized human proteins, respectively (315; 316). In terms of chemical modulators of mitochondrial dynamics, a screen of 7 Trolox- derived small molecules revealed that Trolox ornithylamide hydrochloride could rescue mitochondrial phenotype in fibroblasts from patients with Leigh Syndrome (317). Hence, implementation of HTS approaches may help the discovery of novel modulators of mitochondrial dynamics.

119 Having said that, numerous obstacles including cost and the lack of available algorithms that are capable of automatic morphological feature-extraction of mitochondria have slowed down the use of HTS in the field of mitochondrial dynamics. This lack of automation has resulted in the use of subjective classifications of mitochondrial shape which does not take into account the basal morphological characteristics of the studied cell line, and therefore bias the interpretation of study results (314). In addition, although numerous papers have described sophisticated methodologies to quantify mitochondrial dynamics in different cell types, only a few have quantified the mitochondrial morphology in large-scale models by incorporating multiple replicates (135; 317–323). Hence, the field of mitochondrial dynamics can greatly benefit from the use and development of freely available algorithms capable of feature extraction and analysis of mitochondria morphology.

Lastly and as explained in section 1.3, pathological conditions which specifically affect the cardiovascular system are associated with abnormal fission. We and others have shown that inhibiting mitochondrial fission proteins or activating mitochondrial fusion proteins may have the following beneficial effects: limiting myocardial infarction (7–9); preventing the progression of PHT (10); and promoting stem cell differentiation into cardiomyocytes (206). So far only a handful modulators of mitochondrial fission such as Mdivi-1 (324), P110 (8) and Dynasore (325) have been described; however, these are known to have off-target effects. Hence, the identification of more specific modulators may help improve the therapeutic targeting of mitochondrial fission under pathological conditions.

In this chapter, we examined the efficacy of a publicly-accessible algorithm in

Image J and showed that it can successfully segment and analyse mitochondrial shape and distinguish between groups of cells with different mitochondrial morphology.

120 Based on this algorithm we also performed a cell-based high-throughput screen and identified novel chemical modulators of mitochondrial morphology.

Methods

5.2.1 Cell Culture and Materials

The screen was based on HeLa cells (ATCC-CCL2) due to their use in a separate large-scale screen (135), clear mitochondrial morphology, robust proliferative nature, higher transfection ratio in comparison to cardiac cell lines such as HL-1 and

H9C2. Cells were initially plated in 6-well-plates at the population of 100,000 in

DMEM-Glutamax (ThermoFisher-31966021) and were left to grow for 48 hours (cell were grown on glass cover-slips for confocal microscopy). Cells were then transfected using Xtreme gene 9 vector (Roche, 06365809001) and mitochondrial-targeted RFP

(1:3 ratio). Medium containing the transfection mix was changed after 24 hours and cells were split using trypsin (life-technologies, 25200-056) after 48 hours. These transfected cells were then plated in 384-well-plates (Perkin-Elmer CellCarrier-384

Ultra Microplates Cat. No.:6057300) for the main screen.

5.2.2 Image Acquisition for Algorithm Validation and Parameter Selection

The Bernsen auto local threshold algorithm, available in Image J, was used to detect morphometric descriptors of mitochondrial shape. This method is based on binarisation of captured images and subsequent feature extraction. To assess the efficacy of the algorithm, 60 images of RFP-transfected HeLa cells were taken randomly using a 63x oil immersion objective of SP5 confocal Leica microscope. The images were then assessed for morphological differences and 4 images were selected by three individual readers for each of the following categories:

a) Fragmented,

121 b) Predominantly fragmented, c) Indeterminate, d) Predominantly elongated or, e) Elongated

These groups were then used to select the shape descriptor of interest and assess the sensitivity of the algorithm to distinguish the differences between the groups. The following morphometric shape descriptors were assessed for each of these selected images:

7. “Area in μm2”;

8. “Perimeter in μm”;

9. “Feret’s diameter in μm (defined as the longest line that can be drawn between

any two points within a mitochondrion”;

10. “Roundness (expressed as 4 x Area/π x Major axis2)”; and

11. “Circularity (expressed as 4π x Area/ Perimeter2). Mitochondria with lower

circularity value have an elongated shape.”

To obtain the maximum dynamic range for the HTS assay, the parameter with the highest maximal deviation value (d-value) between the highly elongated and highly fragmented groups was chosen for the assessment of morphology. The sensitivity of the algorithm to correctly identify the mitochondrial morphology was assessed based on the human eye readout and the given range of the morphometric parameters (See

Table 5-1).

5.2.3 Cell Preparation for the Main Screen and Establishing the Dynamic Range

To establish the controls for the dynamic range of the main assay, mt-RFP transfected HeLa cells were plated in either single confocal dishes (4 x 106). Cells were treated with ionomycin (10μM- SIGMA- I0634) or Mdivi-1 (5μM- SIGMA- M019)

122 for a period of 40 minutes (7; 326; 327) ; fixed for 20 minutes in 4% PFA and washed with PBS (10 images were taken per cover-slip using confocal microscopy (N=3)).

Subsequently, the results were compared against the DMSO (0.01%) treated cells. For the actual screen, cells were plated in 384-well plate; divided into 4 equal sections and treated with the drugs from the Prestwick Chemical Library ® (library of 1280 FDA approved small molecules) or the controls with a concentration of 10 μM. In a similar fashion to the previous step, 384-well-plates of the main screen containing the transfected cells were treated for 40 minutes before being fixed in 4% PFA. Study design, plate preparations, cell treatments, image analysis and processing were all performed in collaboration with Dr Janos Kriston-Vizi, Dr Joana Rodrigues Simoes

Da Costa and Dr Robin Ketteler and passed the committee approval of the HTS unit of the LMCB.

5.2.4 Image Acquisition and Processing for the Main Screen

Plates were imaged using the Opera High Content Screening System (Perkin

Elmer). Image acquisition was performed on 384-well-plates using a 60x water immersion objective. 9 fields of view were captured per well (n=4 replicates). Images were processed using the Bernsen algorithm.

5.2.5 Post-test Analysis and Drp1 GTPase Assay of Selected Compounds

Since we were interested in drugs that either caused the least fragmentation/inhibited fragmentation or induced elongation (exhibiting mainly elongated mitochondria) we mainly studied the top 20 drugs and selected 10 compounds based on their novelty; link with mitochondrial function; type of therapeutic effect and reproducibility. However, we also selected 10 drugs from fragmentation end of the spectrum based on the same criteria to further evaluate the

123 sensitivity of the algorithm. In the second part of the analysis, we studied the binding affinity of top 20 hits with mostly elongated mitochondria to the GTPase domain of

Drp1. This work was performed in collaboration with Dr Jessica Holien and Dr Shiang

Yong Lim. from O'Brien Institute Department, St Vincent's Institute of Medical

Research, Melbourne, Australia. For this method, structures were exported from the smiles strings using Chemaxon software. Structures were imported into SybylX2.1.1 and where needed, manually altered. All non-biological salts were removed, and a single 3D concord structure was created using ligprep. All 20 compounds were docked into the GTPase domain of Drp1 using GeomX docking module in Surflex

Sybylx2.1.1. The top 20 docked solutions for each compound were retained and were and were visually analysed. For instance, Zimelidine had hardly any clustering with

Figure 5-1 Example of Docking Cluster for Zimelidine and Phenformin Hydrochloride. (A) shows almost no clusters for Zimelidine after computational docking with GTPase domain of Drp1 (structure of the domain is not shown) whereas (B) shows almost 100% clustering of Phenformin hydrochloride (good and bad docking clusters are shown in red and light grey, respectively).

124 the GTPase domain of Drp1 whereas Phenformin hydrochloride exhibited almost

100% clustering with this domain (See Figure 5-1 which shows docking clusters for these two different drugs). The likelihood rank of each compound interacting with the

GTPase domain of Drp1 was obtained from the addition of their docking score to the relative number of solutions in a cluster to create an arbitrary score. Drugs with the arbitrary score of above 50 were considered as potential binders to the GTPase domain of Drp1.

5.2.6 Statistics

Minitab (V.16) and R-software with custom built script were used to generate the figures and analyse the data. Comparisons between different empirical cumulative distribution functions (ECDF) were performed using the KS test to obtain the relevant p- and d-value. For the main screen, the data from each well of the sixteen 384-well- plates of the main library were normalised based on the per-plate median to eliminate any edge effects. Scoring was performed using the normalised percent inhibition. The

Z-score was obtained by calculating the mean of the plate-replicates and subtracting that value from the overall mean and subsequent division by the standard deviation of the entire library. For all the statistical tests, the p-value ≤ 0.05 was considered significant.

Results

5.3.1 Selection of Shape Descriptor of Interest

The data from each group (shown in Figure 5-2) were collated together and their cumulative frequency distributions were plotted in Figure 5-3. The K.S test between different shape descriptors of fragmented and elongated groups indicated that

125

Figure 5-2 Representative Confocal Images of Mitochondria in Hela Cells for Each Morphological Category. (A), (B) and (C) show the images of cells exhibiting elongated, pre-dominantly elongated or indeterminate mitochondria whereas (E) and (D) illustrate cells showing pre-dominantly fragmented and fragmented mitochondria, respectively.

126

Figure 5-3 Comparison of Frequency Distribution of Shape Descriptors of Different Mitochondrial Population. (A) and (B) indicate the area and perimeter whereas (C), (D) and (E) illustrate the circularity, roundness and Feret’s diameter, respectively. *** denotes P<0.001 and along with the D-values compare Elongated vs. Fragmented (N=4 Images per group).

127 the parameter of roundness (Median fragmented: 0.85 vs. Elongated: 0.5, p< 0.0001,

D=0.502, Figure 5-3D) had the highest d-value between the fragmented and elongated groups in comparison to area (Fragmented: 0.22 vs. Elongated: 0.24, p< 0.0001,

D=0.125, Figure 5-3A), perimeter (Fragmented: 1.68 vs. Elongated: 2.0, p< 0.0001,

D=0.248, Figure 5-3B), Feret’s diameter (Fragmented: 0.68 vs. Elongated: 0.85, p<

0.0001, D=0.286, Figure 5-3E) and circularity (Fragmented: 1.00 vs. Elongated: 0.74, p< 0.0001, D=0.428, Figure 5-3C). Hence, roundness was selected as the parameter of interest because of its high maximal deviation value.

5.3.2 Algorithm Output Comparison to Readout from Trained Human Eye

After selecting the best parameter, we assessed the degree of similarity between the readouts from human eye and algorithm output for roundness. The median range of roundness from the algorithm (shown in Table 5-1) was 0.891 to 0.814

(fragmented), 0.668 to 0.576 (predominantly fragmented), 0.583 to 0.576

(indeterminate), 0.553 to 0.5 (predominantly elongated) and 0.528 to 0.486 for elongated population of mitochondria. 16 out of 20 (80%) images had a roundness median range corresponding to the readout of 3 blind assessors (See Table 5-1 values with stars for roundness median values overlapping with the other groups). The extent of similarity of these readouts can be also observed in the frequency distribution data shown in Figure 5.3. 100% of fragmented and pre-dominantly fragmented images corresponded to the human eye readouts whereas only 75% of the algorithm output resembled those from pre-dominantly elongated or elongated groups. These results indicate a good consistency between the readout of images with trained human eye and the output of the Bernsen algorithm.

128 Table 5-1 Comparison between the output of mitochondrial morphology readouts from the algorithm and human eye. Cell Roundness Human Eye Cell Roundness Human Eye Number Median readout (3 Number Median Readout Value readers) Value (N=3)

1 0.891 Fragmented 11 0.5355 Indeterminate

2 0.870 Fragmented 12* 0.516 Indeterminate

3 0.870 Fragmented 13* 0.553 Pre- Elongated 4 0.814 Fragmented 16 0.522 Pre- Elongated 5 0.668 Pre- 15 0.5 Pre- Fragmented Elongated

6 0.5925 Pre- 16 0.5 Pre- Fragmented Elongated

7 0.5885 Pre- 17* 0.528 Elongated Fragmented

8 0.576 Pre- 18 0.499 Elongated Fragmented

9* 0.583 Indeterminate 19 0.492 Elongated

10 0.575 Indeterminate 20 0.486 Elongated

* Cells which had a median readout range overlapping with another categories range.

129

Figure 5-4 Comparison of Roundness Frequency Distribution in Cells with Different Mitochondrial Population*. The fragmented, elongated, pre-fragmented and pre-elongated population of mitochondria are illustrated by different shades of black, red, green, and blue colour.

*Cells with indeterminate mitochondrial population are omitted from the graph for better presentation of other groups.

130 5.3.3 Establishing the Dynamic Range for the Main Assay

To obtain a positive and negative control with a broad dynamic range we used mitochondrial calcium chelator ionomycin to induce fragmentation and Mdivi-1 to prevent fragmentation. Despite induction of a clear shift in mitochondrial roundness in cells treated with Ionomycin (Median Ionomycin: 0.92 vs DMSO: 0.64, P<0.0001,

D=0.3646, Figure 5-5 C and D), we did not observe any meaningful shift towards

Figure 5-5 Comparison of Roundness Frequency Distribution in Cells After Individual Treatments. (A) shows cells treated with DMSO whereas (B) and (C) indicate cells treated with Mdivi-1 or Ionomycin. The frequency distributions of mitochondrial roundness of images in (A), (B) and (C) are presented in the graph (D). *** and $ denote P<0.05 and P<0.001, respectively (N=3).

131 elongation in cells treated with Mdivi-1 (Mdivi-1: 0.66 vs DMSO: 0.64, P=0.0464,

D=0.0166, see Figure 5-5 A, B, and D). These results indicated that mitochondria in intact HeLa cells cannot be further elongated using Mdivi-1 i.e. Mdivi-1 cannot induce fusion. Hence, due to the lack of any alternative drug capable of inducing fusion the screen was performed using DMSO and ionomycin.

5.3.4 Screen Results of the mt-RFP Transfected HeLa Cells Treated with

Compounds from Prestwick Library

The data obtained from the main screen (See Table 9-1 and 9-2 for the Full list) revealed the presence of negative Z-factors for all four replicates (see Figure 5-6) which indicated a substantial overlap between the roundness of cells treated with either ionomycin or DMSO and a very low dynamic range. However, observing images (See

Figure 5-7) from hits with the highest positive and negative Z-scores (Disulfiram and

Nalfon Fenoprofen calcium salt) revealed marked differences in mitochondrial morphology between the two ends of the Z-score list suggesting that we had successfully identified potential modulators of mitochondrial morphology.

Figure 5-6 Summary of Z'-Factors for Individual Replicates. Z’-Factor value for replicate No.1,2,3 and 4 are shown in (A), (B), (C) and (D), respectively. Blue and red histogram indicate Ionomycin and DMSO, respectively.

132

Figure 5-7 Images from Two Ends of Z-score List. Row (A) shows images from cells treated Disulfiram exhibiting elongated mitochondria whereas (B) shows unhealthy cells exhibiting fragmented mitochondria after treatment with Nalfon Fenoprofen calcium salt.

133 5.3.5 Post-test Analysis and Algorithm Output Verification

A list of drugs that were chosen based on our selection criteria is given in

Table 5-2 and 5-3. Further assessments of these drugs using the human eye revealed that 100% of the drugs in fragmentation (See Table 5-5) end of the Z-score list had fragmented mitochondria. However, only 60% of those drugs on the fusion end (See

Table 5-4) exhibited elongated mitochondria when compared to the human eye readouts. These results were in line with the initial analysis and indicated the difference in the sensitivity of the algorithm to detect cells with fragmented and elongated mitochondria.

Table 5-2 Nominated 10 Drugs from Top 20 Drugs Causing Mitochondrial Elongation/Inhibiting Fragmentation Name of The Mitochondria- Therapeutic Repeatability Ranking Drug related Effect Standard between effects Deviation Top 20 (RSD) Drugs Disulfiram Yes Anti-abuse effect 0.0737 1

Pheniramine Yes Antihistaminic 0.0325 2 maleate

Hydralazine Yes Antihypertensive 0.0543 3 hydrochloride

Mafenide Yes Antibacterial 0.0488 5 hydrochloride

Sulfamethoxazole Yes Antibacterial 0.0562 7

Mefenamic acid Yes Analgesic 0.0286 8

Benoxinate Yes Local Anaesthetic 0.0215 9 hydrochloride Oxaprozin Yes Analgesic 0.0409 14

Phenformin Yes Antidiabetic 0.0526 17 hydrochloride

Minoxidil Yes Antihypertensive 0.0345 19

134 Table 5-3 Nominated 10 Drugs from Top 20 Drugs Causing Mitochondrial Fragmentation/Inhibiting Elongation. Name of The Link with Therapeutic Repeatability Ranking Drug Mitochondria Effect Standard between Deviation Top 20 (RSD) Drugs Fenoprofen Yes Anti- 0.0389 1 Calcium Salt inflammatory

Pyrvinium No Anti-pinworm 0.0318 2 pamoate

Hexestrol Yes Antineoplastic 0.0582 5

Zuclopenthixol Yes Antipsychotic 0.0466 6 dihydrochloride

Eucatropine NO Antiglaucoma 0.213 15 hydrochloride

Tribenoside No Vasoprotective 0.037 16

Ethotoin No Anticonvulsant 0.035 17

Naproxen Yes Anti- 0.226 18 inflammatory

Pridinol NO Anti-Parkinson 0.163 19 methanesulfonate

Irsogladine No Antiulcer 0.0318 20 maleate

135 Table 5-4 Re-Scoring the Images Using Human Eye for Drugs Causing Mitochondrial Elongation/Inhibiting Fragmentation

Human Eye Readout of Name of the drug No. and (%) of Images with Different Mitochondrial Population (Average of 2 Readers): Elongated Indeterminate Fragmented Blank Images Disulfiram* 10 14 10.5 1.5 (27.8%) (38.9%) (29.2%) (4.2%)

Pheniramine 21.5 11 2.5 1 able

T maleate (59.7%) (30.6%) (6.9%) (2.8%)

Hydralazine 16 7.5 9 3.5 hydrochloride (44.4%) (20.8%) (25.0%) (9.7%)

Mafenide 6 8.5 17 4.5 hydrochloride* (16.7%) (23.6%) (47.2%) (12.5%)

Sulfamethoxazole 6 14.5 11.5 4 valuein Hits Main the - * (16.7%) (40.3%) (31.9%) (11.1%)

Mefenamic acid 20 11 4.5 0.5 (55.6%) (30.6%) (12.5%) (1.4%)

Benoxinate 15.5 14.5 5.5 0.5 hydrochloride (43.1%) (40.3%) (15.3%) (1.4%)

Oxaprozin 29.5 5 0 1.5 (81.9%) (13.9%) (0.0%) (4.2%)

Phenformin 12 8 12 4 hydrochloride* (33.3%) (22.2%) (33.3%) (11.1%)

rugs are ordered according ordered Z are rugs their to D Minoxidil 14.5 10 8.5 3 (40.3%) (27.8%) (23.6%) (8.3%)

*Drugs with images that did not exhibit fused population of mitochondria.

136 Table 5-5 Re-Scoring the Images Using Human Eye for the Drugs Causing Fragmentation/Inhibiting Elongation.

Human Eye Readout of No. and (%) of Images with Different Mitochondrial Name of the Drug Population (Average of 2 Readers): Elongated Indeterminate Fragmented Blank Images Nalfon Fenoprofen 0 0 12 24 (0%) (0%) (33.3%) (66.7%)

Pyrvinium pamoate 0 0 17 19 (0%) (0%) (47.2%) (48.7%)

able T Hexestrol 1 4 31 0 (2.8%) (11.1%) (86.1%) (0%)

Zuclopenthixol 10 5 20 1 dihydrochloride (27.8%) (13.9%) (55.6%) (2.8%)

Eucatropine 2.5 1.5 7 25 valuein Hits Main the

- hydrochloride (6.9%) (4.2%) (19.4%) (69.4%)

Tribenoside 11.5 5.5 18 1 (31.9%) (15.3%) (50%) (2.8%)

Ethotoin 2.5 8 23 2.5 (6.9%) (22.2%) (63.9%) (6.9%)

Naproxen 1.5 2.5 9.5 22.5 (4.2%) (6.9%) (26.4%) (62.5%)

Pridinol 5 9.5 8 13.5 methanesulfonate (13.9%) (26.4%) (22.2%) (37.5%)

rugs are ordered according ordered Z are rugs their to D

Irsogladine 2.5 7 23 24.5 (6.9%) (19.4%) (63.9%) (68.1%)

137 5.3.6 Computational Docking for the GTPase Domain of Drp1

To further validate the hits, the top 20 drugs from the end of the Z-score list with abundantly elongated mitochondria were assessed in terms of their degree of docking to the GTPase domain of Drp1. All 20 compounds could dock into the GTPase domain of Drp1. However, visual assessment suggested that only some of these drugs had a “real” binding affinity to the GTPase domain. Assessment of the clustering percentage and docking score shown in Table 5-6 revealed that only half of these drugs were potential binders to the GTPase domain of Drp1. Five of these drugs including

Minoxidil, Hydralazine hydrochloride, Benoxinate hydrochloride, Oxaprozin and

Pheniramine maleate were shown earlier to have caused extensive elongation and hence were selected for further analysis. Together, these data revealed that 50% of the drugs that could either inhibit fragmentation or induce elongation could dock to the

GTPase domain of Drp1.

Discussion

In this chapter, we initially showed the importance of mitochondrial shape descriptors for morphological analysis and determined the mitochondrial roundness as the most sensitive parameter for our screen. Using a validated algorithm, we subjected mt-RFP transfected HeLa cells to small molecules from Prestwick chemical library and identified potential hits capable of inducing elongation/inhibiting fragmentation.

Lastly, we re-evaluated the hits using the human eye readout and their docking ability to bind to the GTPase domain of Drp1 which helped us select five potential candidates for further studies.

The automatic analysis of mitochondrial morphology has gained grounds in recent years and unlike manual scoring, has reduced both the time required for

138 Table 5-6 The Docking Scores of Top 20 Drugs Causing Elongation/Inhibiting Fragmentation Name of the Drug Therapeutic Docking cluster Total Effect score percentage Phenformin Antidiabetic 5.81 90 95.81 hydrochloride* Minoxidil*$ Anti-alopecia 5.16 90 95.16

Phenelzine sulfate* Antidepressant 4.63 90 94.63 Hydralazine Antihypertensive hydrochloride*$ 4.58 80 84.58

Glipizide* Antidiabetic 7.39 75 82.39

Nitrofurantoin* Antibacterial 5.03 75 80.03 Benoxinate Local hydrochloride*$ anaesthetic 7.23 50 57.23

Alverine citrate salt* Anti-spastic 6.99 50 56.99 Metoclopramide monohydrochloride* Antiemetic 6.67 50 56.67

Oxaprozin*$ Analgesic 6.09 50 56.09 Pheniramine maleate*$ Antihistaminic 4.29 50 54.29

Mefexamide hydrochloride CNS Stimulant 6.22 25 31.22

Tacrine hydrochloride CNS Stimulant 4.12 25 29.12

Mafenide hydrochloride Antibacterial 4.00 25 29.00

Diazoxide Antidiuretic 3.73 25 28.73 Disulfiram Antabuse effect 3.97 5 8.97

Sulfamethoxazole Antibacterial 6.22 0.00 6.22 Mefenamic acid Analgesic 4.53 0.00 4.53

Zimelidine dihydrochloride Antidepressant 4.23 0.00 4.23 monohydrate

Lynestrenol Contraceptive 4.23 0.00 4.23 * Drugs with potential binding affinity to the Drp1 GTPase domain.

$ Drugs with images which showed mostly fused mitochondria (See Table 5-2)

139 morphological identification of mitochondrial fragments and relative subjectivity in assessment (317; 323; 328). The most important components of methods described for the analysis of mitochondria morphology are the thresholding algorithm and the morphological parameter of interest (329). The only known large- scale screen concerned with mitochondrial morphology has been the investigation by

Norton et al. (2014) where they assessed the effects of 18,255 siRNA on human genes in HeLa cells. Interestingly and consistent with our methodology, they used a single parameter known as aspect ratio (“roundness is the inverse of aspect ratio”) and were successfully able to detect the morphological effects of different siRNAs (135).

However, they used Cellomics vHCS Scan commercial package, which compared to our method, is not an open source and hence is not publicly available. Although a single parameter approach has been used in many studies dealing with mitochondrial morphology, mitochondrial shape factors can be independent of each other (See

Chapter 4). Hence the usage of multi-parametric approach may greatly help in the determination of the type of effect elicited by the given drug (318). In line with this fact, Koopman et al. (2015) showed that the multi-parametric assessment of mitochondrial morphology can be optimised to select the best derivate of Trolox variants in reversing the morphological abnormalities seen in fibroblasts from patients with Leigh syndrome. In addition, they showed higher algorithm sensitivity to correctly identify true hits in comparison to our approach (312). This can be partially explained in our study by the lack of any known inducer of mitochondrial fusion to obtain the optimal dynamic range. However, and despite advances in the multi- parametric assessment of mitochondrial morphology, little is known about their usefulness in large-scale screenings (318). In addition, multi-morphometric parameter usage for hit identification requires advanced statistical and computational expertise

140 which are currently available in only a handful laboratories and are not widely accessible. Furthermore, it is also important to mention that as previously discussed, mitochondria are 3D entities and hence HTS can additionally benefit from the 3D evaluation of mitochondrial shape (330). However, 3D image acquisition and processing are time-consuming and add another layer of complexity to the involved computational examinations.

As discussed earlier and in chapter 1, inhibition of fission has been shown to elicit therapeutic benefits in numerous pathological conditions and especially in the setting of cardioprotection following IRI (7; 9). Fission is highly dependent on the function of Drp1. The GTPase domain of Drp1 allows its oligomerisation and helix formation upon translocation to the mitochondria (324; 331). Drugs such as Mdivi-1 have been shown to inhibit Drp1 self-assembly to mitochondria. However, this mechanism has been shown to be independent of the GTPase function of Drp1 (324).

Refining the identified hits using post-screen analysis revealed the presence of 5 drugs that either inhibited fragmentation or induced elongation. These drugs could also specifically bind to the GTPase domain of Drp1. Between the five drugs, hydralazine and minoxidil had the highest binding affinity to the GTPase domain of Drp1. These two drugs are anti-hypertensive and are known to elicit potential cardioprotective effects (332; 333). Minoxidil, which is a potent mitochondrial K+ opener, can prevent ischaemic cell death via a similar action to those from Diazoxide by preserving mitochondrial membrane potential and morphology under IRI (332; 334; 335).

Similarly, hydralazine has also been documented to confer cardioprotection following in-vivo IRI in rats, however, its exact mechanism on cardioprotection is unknown

(333). In contrast, pheniramine, an antihistaminic small molecule, does not elicit protection against simulated ischaemia in isolated rat ventricular myocytes (336).

141 Likewise, benoxinate hydrochloride, which is often used for optical anaesthesia, has been reported to have lower cardiotoxicity effects in comparison to its equivalent anaesthetic drugs but has not been studied in the setting of cardioprotection (337).

Lastly, oxaprozin may have an adverse cardiovascular effect because of being from a non-steroidal anti-inflammatory family of drugs and hence its use may be limited in the setting of cardioprotection (338). Overall, these drugs may have valuable therapeutic benefits since they exhibit promising characteristics in terms of modulating the Drp1 activity. These drugs were shown to interact with Drp1 GTPase domain but whether they affect, other members of mitochondrial fission as well as members of mitochondrial fusion machinery remains an open question.

Conclusion

In summary, using an open source algorithm we could threshold and extract morphological features of mitochondria. We addressed the sensitivity of the algorithm and assessed its performance in identifying novel small molecules modulators available in Prestwick chemical library. We identified several hits that were potentially capable of inhibiting Drp1 function to trigger fission and one of these hits was selected to be the focus of the next chapter of this project.

142 Chapter 6. Mitochondrial Dynamics and Hydralazine Induced Cardioprotection

Introduction

Cardiac homeostasis depends heavily on the maintenance of mitochondrial dynamics, and alterations in fusion and fission events can result in cardiovascular dysfunction and disease (26; 156; 198; 202; 205). The concept of mitochondrial dynamics incorporates inter-mitochondrial communication and changes in physical morphology of mitochondria by fission and fusion processes (17; 97). While much attention has been paid to the alteration of mitochondrial fission and fusion proteins and subsequent changes of mitochondrial morphology, the exact relevance of mitochondrial communication in terms of fusion events in adult heart is not fully appreciated. Pathological conditions such as hypoxia are known to reduce the occurrence of mitochondrial fusion events in H9C2 myoblast cell lines (92), but whether the same concept applies to the adult cardiomyocytes and whether mitochondrial fusion can be targeted by a therapeutic approach in the setting of cardiac-related IRI is currently unknown.

Therefore, novel therapeutics are required to improve clinical outcomes of

CHD patients following acute IRI (339). The alteration of mitochondrial dynamics by inhibition of mitochondrial fission has been shown to confer cardioprotection following IRI (7; 9). Using the HTS approach described in Chapter 5, we identified hydralazine to be a novel inhibitor of mitochondrial fission. Pharmacological benefits of hydralazine are extended from treatment of T-cell lymphoma to the inhibition of atherosclerosis-related neovascularisation. However, hydralazine is mainly used as an anti-hypertensive drug, particularly to manage the symptoms of pre-eclampsia (340–

342). Hydralazine administration in combination with isosorbide dinitrate is also a

143 common treatment for African American patients suffering from reduced ejection fraction HF (343). These therapeutic effects of hydralazine are imposed by its direct vasodilatory action which involves arterial vascular dilatation and a compensatory decrease in peripheral vascular resistance which in turn increases the heart rate and cardiac output (344). Moreover, the acute administration of hydralazine has been shown to elicit cardioprotection and reduce infarct size following IRI in experimental studies, however, the exact cardioprotective mechanisms of hydralazine in this setting is currently undetermined (333). Possible cardioprotective effects of hydralazine may be attributed to its actions to stabilise HIF1-α, upregulate cGMP expression, inhibit the IP3-related SR release of Ca2+ in vascular smooth muscle cells and scavenge intracellular ROS (345–351). In this chapter, we investigate whether the cardioprotective effect of hydralazine can be explained by its effect, given that in the previous chapter, we showed that hydralazine can modulate mitochondrial morphology and is able to bind to the GTPase domain of Drp1.

In this chapter, we demonstrate the existence of mitochondrial fusion events in adult mouse cardiomyocytes and study the frequency of these events in different subpopulations of adult cardiac mitochondria. Additionally, we show that the mitochondrial fusion events are severely disturbed under the influence of HR and demonstrate the inhibition of this effect by hydralazine treatment. Finally, we show that the preservation of mitochondrial dynamics by hydralazine prevents the occurrence of cell death and reduces the infarction in ex-vivo Langendorff IRI model thereby demonstrating a novel mechanism for hydralazine-induced cardioprotection.

144 Methods

6.2.1 Animals and Cell Culture

Animal work was conducted in compliance with the Singapore National

Advisory Committee for Laboratory Animal Research (NACLAR) guidelines, in collaboration with Dr Sang Bing Ong, Miss Kwek Xiu Yi and Mrs Khairunnisa Binte

Katwadi from Duke-National University of Singapore. Female 10-16 weeks old

Dendra mice (B6;129S-Gt(ROSA)26Sortm1(CAG-COX8A/Dendra2) Dcc expressing mitochondrial targeted Dendra 2 were purchased from Jackson Laboratory and were used for the assessment of mitochondrial fusion events and morphology. Age-matched female C57/BL6 were used for the rest of the experiments. Cell line work was performed on HeLa cells which were transfected according to the protocol explained in section 5.2.1. For the cell culture of this chapter, cells were grown on cover-slips that were placed in 6 well plates with the initial density of 80,000. All Materials were purchased from Sigma unless otherwise is stated.

6.2.2 Dose-Response Determination of Hydralazine Concentration

After transfection, cells were treated with either 1, 10 or 100 μM hydralazine for 40 minutes and were compared individually versus the time-matched DH2O vehicle control (Veh. CT.) (N=3). For imaging, coverslips were placed inside confocal rings and were loaded with Tyrode’s buffer (NaCl (137mM), KCL (5mM), MgCl2 (0.4mM),

CaCl2 (1mM), D-Glucose (10mM), Na HEPES (10mM) at pH 7.4.) containing the relevant concentration of each drug. For each individual cover-slip, 8 random images were acquired using the x40 objective of a Leica xSP5 microscope in a blinded fashion and analysed using Bernsen algorithm (Kernel 5) of Image J. Mitochondrial roundness was used for the evaluation of morphological differences.

145 6.2.3 Characterisation of H2O2 Induced Fragmentation and Membrane Potential

Depolarisation and Hydralazine Impact on H2O2 Treated Cells

For characterising the effect of H2O2 induced fragmentation, cells were treated for 30 or 60 minutes with 3.3mM H2O2 (dose was based on a previous report by

Reichert group (122)) and were compared against Veh. CT. (N=3). For evaluating the effect of hydralazine, cells were treated with either Veh. CT. (N=5) or hydralazine

(1uM) for 40 minutes. Subsequently, cells were treated for 60 minutes with H2O2 in the presence of either Veh. CT. or hydralazine (N=6). The same methodology, explained in section 6.2.2, was used to capture images from each of the mentioned groups and the extent of fragmentation induced by H2O2 was measured by quantifying mitochondrial roundness. Mitochondrial membrane potential was assessed by incubating the cells in rhodamine 123 (2 μM) for the last 30 minutes of individual treatments. Mitochondrial membrane potential was analysed using the Otsu algorithm available in Image J software and via the corrected total cell fluorescence (CTCF) method (352).

6.2.4 Cardiac Cell Isolation

Extracted hearts were cannulated and perfused using the “perfusion” buffer containing NaCl (113 mM), KCl (4.7mM), KH2PO4 (0.6mM), Na2HPO4(0.6mM),

MgSO4-7H2O (1.2mM), NaHCO3 (12mM), KHCO3 (10mM), HEPES Na Salt

(0.922mM), Taurine (30mM), BDM (10mM) and Glucose (5.5mM) for a period of 5 minutes. Tissue lysis was conducted by perfusing the hearts with liberase (5mg/ml,

Roche-05401127001) for 3 minutes. Isolated cardiomyocytes were then serially incubated in the perfusion buffer containing 10% FBS and 200 µM, 400 µM or 900

µM CaCl2 for 10 minutes. Pellet of isolated cardiomyocytes was finally re-suspended

146 in M199 solution containing L-carnitine (2mM), creatine (5 mM), Taurine (5mM) and blebbistatin (25µM) and plated on laminin-coated wells.

6.2.5 Hypoxia Reoxygenation (HR) Model

Isolated cardiomyocytes were divided into three groups and treated according to the protocol illustrated in Figure 6-1. Cells in normoxic time control (CT.) group were incubated in the buffer containing KH2PO4 (0.5mM), NaHCO3 (5mM),

MgCL2.6H2O (0.6mM), Na Hepes (12.5mM), NaCl (97.60mM), KCL (2.9mM), D-

Glucose (10mM), Na-Pyruvate (2mM) and CaCl2 (1.26mM) at pH 7.4 for 60 minutes.

Cardiomyocytes in the other two groups received either the Veh. CT. or hydralazine

15 minutes before being treated with hypoxic buffer containing KH2PO4 (0.5mM),

NaHCO3 (5mM), MgCL2.6H2O (0.6mM), Na Hepes (12.5mM), NaCl (74mM), KCl

(16mM), Na-Lactate (20mM) and CaCl2 (1.26mM) at pH 6.2 in a sealed hypoxic chamber for 30 minutes. The hypoxic buffer was subsequently replaced with normoxic buffer for 15 minutes before further downstream procedures.

Figure 6-1 HR Protocol in Isolated Cardiomyocytes. This diagram illustrates the HR protocol and the treatment duration for each individual group.

147 6.2.6 Evaluation of Mitochondrial Fusion Assay and Mitochondrial Morphology

After the termination of each protocol shown in Figure 6-1, isolated cardiomyocytes from Dendra 2 mice were imaged in the Tyrode’s buffer. Images were taken blindly using a Nikon A1 confocal equipped with a live cell humidity-controlled imaging chamber at 37 °C (5% CO2). Each well was divided into four regions and if present, cells with fragmented mitochondria were prioritised for imaging in each region. In the absence of cells with fragmented mitochondria, cells with normal mitochondrial morphology were imaged. For each individual group (N=5 for Veh. CT and Normoxic time CT. and N=4 for hydralazine) at least 20 cardiomyocytes were imaged at every 4 minutes for a total period of 16 minutes. For each of these cells, three regions of interest with a fixed size of 1.24 µM2 targeting individual mitochondrion in IMF, SSM or PN regions (See Figure 6-10 and Section 3.2.5 for the relevant terminology) were activated using a 405-nm laser. Images were stacked and aligned using the TurboReg plugin of Image J. Fusion events were counted for single mitochondrion if there was a clear transfer of signal from two neighbouring mitochondria, as shown in Figure, 6-2 during the 16 minutes period. Fluorescent decay within each region of interest was calculated using the integrated density parameter

(measured using Image J) at every 4 minutes (16 minutes in total). Mitochondrial morphology was also crudely analysed based on the number of cells presenting either fragmented or elongated mitochondrial population. Confocal imaging was performed in collaboration with Michelle Tan Guet Khim from Department of Clinical

Translational Research (Singapore General Hospital).

148 6.2.7 Cell Death

After the HR protocol (See Figure 6-1) cells were incubated in 1µL/mL of PI solution

(P4864 sigma) in Tyrode’s buffer for 1 minute. Images were blindly acquired using a

10X dry objective of a Leica DMi8 microscope. For each treatment (N=5 animals per group) 5 images were randomly taken and the PI-positive cells were counted using

Image J.

Figure 6-2 Tracking Mitochondrial Fusion Dynamics in Adult Cardiomyocytes. (A) Depicts series of images from left to right which show the photo-activation and subsequent fusion from the mitochondrion marked by red arrow to mitochondria shown by white arrows. (B) Shows the photo-activation and spread of dendra2 probe in a single channel at each time point across the length of the line drawn on the image. (C) illustrates the spread of signal across the line drawn in (B) at each time point and (Star show the occurrence of fusion events)

149 6.2.8 Ex-Vivo Langendorff Infarct Model

Hearts were extracted from normal female C57/BL6 (n=9) mice and were cannulated and treated according to the protocol shown in Figure 6-3 using the modified Krebs-Henseleit buffer containing NaCl (118.5 mM), NaHCO3 (25.0 mM),

D-glucose (11.0 mM), KCl (4.7 mM), MgSO4.7H2O (1.2 mM), KH2PO4 (1.2 mM), and

CaCl2.2H2O (2.5 mM) at pH 7.4 (Gassed with Carbogen). Hearts in the Veh. CT or hydralazine group were stabilised by the perfusion of Krebs buffer for 15 minutes.

Subsequently, they received either the Veh. CT or hydralazine, based on initial treatment, for another 15 minutes before undergoing 35 minutes of ischaemia. The hearts were then perfused for 60 minutes in the presence of either Veh. CT or hydralazine. At the end of each experiment, hearts were excised and frozen overnight.

Subsequently, hearts underwent the TTC treatment (see section 3.2.2) the next day before being scanned and analysed in Image-J. This section of the work was performed in collaboration with Dr Kroekkiat Chinda from Singapore National Heart

Centre/Duke-NUS.

Figure 6-3 Langendorff Protocol for the Assessment of Infarct Size in Hearts Treated with Hydralazine or Veh. CT. This diagram illustrates the Langendorff protocol and the treatment durations for groups receiving either hydralazine or Veh. CT.

150 6.2.9 Hydralazine Effect on the GTPase Activity of His-Tagged Drp1 and Real-

time Assessment of Hydralazine Binding to the His-tagged Drp1 Protein

Using Surface Plasmon Resonance (SPR).

For His-tagged Drp1 production, bacterial recombinant cDNA of human Drp1

(N-term his6) protein (kindly supplied from Dr Michael T Ryan) cloned into pQE-30 vector and transformed in Rosetta (DE3) competent cells (Novagen, Merck Millipore).

Cells were then grown in 100 µg/mL ampicillin at 37°C, 120 rpm to A600 = 0.6. They were then incubated at 16°C for 18 hours in the presence of 0.5 mM IPTG. Cells were subsequently harvested by centrifugation at 3,500 rpm (20 minutes) and lysed using a buffer made of 50 mM Tris.HCl (pH 7.3), 0.5 M NaCl, 50 mM imidazole, 5% glycerol,

2 mM β-mercaptoethanol, 0.1 mM LEUPEP, 0.1 mM AEBSF and 1 mM benzaminidium chloride. Cell lysates were sonicated and centrifuged at 20,000 rpm for 30 minutes before being loaded onto a 5 mL Nickel Chelating Sepharose Fast Flow column (GE Healthcare, Buckinghamshire, UK). The column was rinsed with wash buffer (50 mM Tris.HCl (pH 7.6), 150 mM NaCl, 10% glycerol and 2 mM β- mercaptoethanol), and Drp1 protein was eluted with wash buffer supplemented with

400 mM imidazole. Eluted proteins were equilibrated with 50 mM Tris.HCl (pH 7.6),

150 mM NaCl, 10% glycerol, 2 mM TCEP using a PD-10 desalting column. GTPase activity of Drp1 was assessed by incubating 100ng of the His-tagged Drp1 with different dosages of hydralazine hydrochloride (Sigma-Aldrich) for 30 minutes at

37oC using the GTPase assay kit (Novus Biologicals, CO, USA).

SPR was performed using the Biacore T200 machine. For this experiment, the human His-tagged Drp1 protein was captured on flow cell 2 of a Xantec NiHC 1200 chip at 30µL/min for 5mins using immobilisation buffer (10mM HEPES (0.15M NaCl,

50µM EDTA, 0.005% Tween20, pH 7.5)). The protein captured well on the Ni2+ with

151 a final amount on the surface of 10371 resonance unit (RU). Flow cell 1 was blocked for use as a reference. The same method was used to capture Drp1 onto flow cell 4, with flow cell 3 as a reference. A kinetic experiment was conducted using hydrazine hydrochloride in a 2-fold concentration series from 500µM to 0.1 µM in HBS-EP+ buffer. The experiment was run in duplicate, twice against all four flow cells. The bottom two concentrations were removed from the analysis as they showed no binding.

To confirm specific binding, hydrazine hydrochloride was also passed against an unrelated protein to Drp1, PA2G4. These sections were performed in collaboration with Dr Jessica Holien, Dr Shiang Yong Lim, and Miss Naomi XY Ling and Dr

Jonathan S Oakhill from O'Brien Institute Department, St Vincent's Institute of

Medical Research, Melbourne, Australia.

6.2.10 Statistics

Analysis and statistics were performed using Graph Pad prism and Mini-Tab software. For dose-response, Kruskal Wallis test and KS test was used to compare the distributions of individual treatments with vehicle control. The D-value of each test was used to select the best dosage. Man -Whitney U test, unpaired t-test or One-way

ANOVA were used to analyse different treatment groups in the rest of the experiments.

P value ≤ 0.05 was considered significant.

Results

6.3.1 Dose-Response Assessment of Mitochondrial Morphology in Cells Treated

with Hydralazine

Based on the concentration used in our screen and a previous report (349) we assessed the effect of three different dosages of hydralazine including 1, 10 and 100

µM on mitochondrial morphology (See Figure 6-4). Kruskal Wallis test indicated a

152 significant difference between 1 to 100 µM dosages (See Figure 6-4 D and Table 6.1).

Figure 6-4 Response of Mitochondrial Morphology to Different Dosages of Hydralazine. (A), (B) and (C) show pairwise analysis of frequency distribution of different treatment groups (N=3) incubated with 1, 10 and 100 µM hydralazine over a period of 40 minutes, respectively. (D) shows the analysis output of Kruskal Wallis test that covers the between-group differences. $ denote P<0.05 vs. 1µM hydralazine whereas ** and *** denote P<0.01 and P<0.001 vs. Veh. CT, respectively (N=3).

153 Individual frequency distribution KS tests indicated that only 1 µM (Figure 6-4A) hydralazine could induce a slight shift towards the lower degree of roundness between the three dosages whereas 10 (Figure 6-4 B) and 100 µM (Figure 6-4 C) concentration had a negligible effect or increased mitochondrial roundness, respectively (See Table

6-1 for the relevant statistics). Hence, we selected 1 µM concentration for further studies of mitochondrial morphology.

Table 6-1 Statistical Parameters of the Effect of Different Dosages of Hydralazine on Mitochondrial Roundness

Treatment Name/ Veh. CT. 1µM 10µM 100µM Statistical Hydralazine Hydralazine Hydralazine parameter

Median 0.5 0.5 0.5 0.5

Mean 0.513 0.507 0.52 0.522 $

Std. Error 0.0007 0.0007 0.0007 0.0007

D and P. D=0.024 D=0.011 D=0.023 Value P< 0.0001 P=0.008 P< 0.0001 Vs. Veh. CT

$ refers to Kruskal Wallis P<0.0216 of 1 µM vs. 100µM Hydralazine

6.3.2 Hydralazine Protects Against H2O2 induced Fragmentation

Oxidative stress caused by H2O2 has been shown to be capable of inducing mitochondrial fragmentation (353). Using a previously described dosage of H2O2

(122), we showed that the treatment of HeLa cells for 30 minutes was enough to cause some degree of fragmentation (Median H2O2.:0.5 vs. Veh. CT:0.5, P<0.0001,

D=0.075, See Figure 6-5 A and Figure 6-6 A and B) and a non-significant reduction in membrane potential (See Figure 6-5 C). Although 60 minutes of H2O2 treatment

154 induced the same level of decrease in mitochondrial membrane potential (See Figure6-

5 C), the extent of fragmentation was more pronounced (H2O2.:0.58 Vs. Veh. CT:0.5,

Figure 6-5 Effect of Different H2O2 Incubation Time on Mitochondrial Morphology and Membrane Potential.

(A) and (B) indicate the H2O2 induced fragmentation (N=3), measured by the increase in mitochondria roundness, after 30 and 60 minutes, respectively. In (C), left panel illustrates the alteration of mitochondrial membrane potential in response to H2O2 or Veh. CT. treatment over different time points. The representative images of each of these group are shown on the right panel. *** denotes vs. Veh. CT. P<0.001 (N=3).

155 P<0.0001, D=0.169, See Figure 6-5 B and Figure 6-6 C and D). Taken together, these data revealed that the treatment of HeLa cells with H2O2 for a period of 60 minutes was enough to trigger marked mitochondrial fragmentation.

Figure 6-6 Effect of Different H2O2 Incubation Time on Mitochondrial Morphology.

(A) and (B) show Veh. CT. and H2O2 treated cells, respectively, after 30 minutes incubation period whereas (C) and (D) show the same treatments after 60 minutes incubation period.

156 Hydralazine is a known ROS scavenger (351) and based on this feature we investigated its effects in the setting of H2O2-induced mitochondrial fragmentation.

Indeed, hydralazine significantly prevented the decrease in mitochondrial membrane potential (Mean ± S.E. hydralazine: 228.3 ± 24.12 Vs. 134.6 ± 6.045, P=0.0073, See

Figure 6-7 B). Hydralazine-treated cells exhibited a lower degree of fragmentation in comparison to the Veh. CT. (Hydralazine:0.5 ± Vs. Veh. CT:0.5, P<0.0001, D=0.055,

See Figure 6-6A and 6-7 A and B). Overall, these data suggested that hydralazine prevented the occurrence of H2O2-induced mitochondrial fragmentation and preserved mitochondrial membrane potential.

Figure 6-7 Hydralazine Induced Preservation of Mitochondrial Shape and Membrane Potential after H2O2 Insult. (A) illustrates the extent of mitochondrial fragmentation, measured by the alteration in mitochondrial roundness, in hydralazine and Veh. CT. treated cells after H2O2 treatment. In (B), the left panel illustrates the alteration of mitochondrial membrane potential in response to H2O2 in Veh. CT. or Hydralazine treated cells. The representative images of each of these groups are shown on the right panel. ** and *** denote P<0.01 and P<0.001 vs. Veh. CT, respectively (N=5 Veh. CT and N=6 Hydralazine).

157

Figure 6-8 Preservation of Mitochondrial Morphology by Hydralazine in H2O2 Treated Cells Column (A) and (B) indicate Veh. CT. and hydralazine treated cells, respectively.

158 6.3.3 Hydralazine Preserves Mitochondrial Dynamics in Cardiomyocytes

Following HR Insult

The relevance of mitochondrial dynamics to adult cardiac myocytes is not fully understood. Here, we initially studied the effect of hydralazine on mitochondrial morphology after HR insult in the presence or absence of hydralazine. Extensive

Figure 6-9 Hydralazine Induced Preservation of Mitochondrial Morphology in HR Treated Cardiomyocytes (A), (B) and (C) indicate the representative images of cells in Normoxic time CT. (N=5), Veh. CT. (N=5) and hydralazine (N=4) groups. The percentages of cells with fragmented mitochondria in each group are shown in (D). (no Fragmentation was observed in Normoxic time CT group)

$ and *** denote P<0.05 and P<0.001 vs. Veh. CT. and Normoxic Time CT, respectively (N=5 for Normoxic Time CT and Veh. CT and N=4 for Hydralazine).

159 fragmentation was observed in Veh. CT. cells receiving HR (Figure 6-9 B) in comparison to hydralazine (Figure 6-9 C). Analysis of data suggested that there was a significant difference between Veh. CT and hydralazine groups (Mean ± S.E. Veh.

CT: 53.93% ± 11.38 Vs. hydralazine: 16.88% ± 6.71, P<0.031, see Figure 6-9 D) in terms of images with cardiomyocytes possessing fragmented mitochondria. No significant difference was observed between hydralazine and normoxic time CT.

Therefore, these results indicated that hydralazine treatment prior to the HR prevents the occurrence of mitochondrial fragmentation in adult cardiomyocytes.

Having examined the effect of hydralazine in the setting of HR, we then investigated the nature of fusion events in three different subpopulations of adult cardiac mitochondria. Time-lapse imaging of photo-activated mitochondria from adult cardiomyocytes expressing mitochondrial targeted Dendra-2 indicated that mitochondrial fusion events were present in all mitochondrial subpopulations of adult cardiomyocytes (See Video S1 and Figure 6-10 white arrows) in normoxic time CT.

The fusion events were mostly present between mitochondria positioned longitudinally to the photo-activated mitochondria and occasionally to the mitochondria positioned transversely between the myofibrils. Analysis of fusion events frequency in untreated normoxic time CT. cardiomyocytes showed that fusion events were more abundant in IMF mitochondria in comparison to SSM and PN mitochondria (Mean ± S.E. IMF:78.60% ± 4.67 Vs. PN: 63.80% ± 1.99 Vs. SSM:

64.00% ± 3.00, overall ANOVA p=0.014, See Figure 6-11 A). ; however, no significant difference was present between PN and SSM mitochondria. Despite the presence of unalike fluorescence decay between individual subpopulation of mitochondria, none of these differences was statistically significant (Figure 6-11 B).

Overall, these data imply that the mitochondrial dynamics in the form fusion events

160 are present in adult cardiomyocytes and their frequency differ according to the subpopulation of adult cardiac mitochondria.

Figure 6-10 Fusion Events after Photo-activation in Normoxic Time CT. Group. Top row indicates the photo-activated mitochondria in red channel whereas the bottom row indicates both red and green channels. The region of interest for individual subsets of mitochondria are illustrated using the yellow circles. White arrows indicate the

occurrence of fusion events.

161

Figure 6-11 Mitochondrial Fusion Characteristics in Untreated (Normoxic Time CT.) Cardiomyocytes. (A) Shows total fusion events for each population of cardiac mitochondria over the period of 16 minutes. The fluorescence decay patterns for individual subtypes of adult cardiac mitochondria are shown in (B). The fluorescence decay is presented as % reduction in the fluorescence intensity from an earlier time point to the next time point. * denotes P<0.05 vs. IMF (N=5 per group). As mentioned earlier, the induction of HR triggered extensive fragmentation which was reversed by the action of hydralazine. HR-mediated fragmentation also hampered the occurrence of mitochondrial fusion dynamics since cells with fragmented mitochondria exhibited no fusion events (See Video S2 and Figure 6-12).

Interestingly, the protective effect of hydralazine was not limited to mitochondrial morphology since it also rescued mitochondrial fusion events in cardiomyocytes that underwent HR treatment (See Video S3 and Figure 6-13).

162

Figure 6-12 Fusion Events in Veh. CT. Group after HR. Top row indicates the photo-activated mitochondria in red channel whereas the bottom row indicates both red and green channels. The region of interest for individual subsets of mitochondria are illustrated using the yellow circles.

163

Figure 6-13 Fusion Events in Hydralazine Group after HR. The top row indicates the photo-activated mitochondria in red channel whereas the bottom row indicates both red and green channels. The region of interest for individual subsets of mitochondria are illustrated using the yellow circles. White arrows indicate the occurrence of fusion events. Having observed the protective effect of hydralazine on mitochondrial fusion events, we then sought to examine whether this process occurs in a subset-specific manner. Despite the presence of trend towards the preservation of fusion events in all subpopulation of mitochondria under the influence of hydralazine (See Figure 6-14 A,

B, and C), only the fusion events of IMF mitochondria were preserved by the action of hydralazine (Mean ± S.E. normoxic time CT.: 78.60% ± 4.67 Vs. Veh. CT.: 24.80

± 5.80 Vs. Hydralazine: 57.25 ± 7.28, overall ANOVA p<0.0001, See Figure 6-14 A).

164 Fluorescence decays followed a similar pattern to the mitochondrial fusion events (See

Figure 6-14 bottom row), however, no significant differences were observed between treatment groups for all mitochondrial subpopulations. Therefore, hydralazine rescues mitochondrial fusion events in the adult cardiomyocytes via a subpopulation specific manner.

Figure 6-14 Mitochondrial Fusion Events for Individual Subpopulations after Each Treatment. Column (A), (B) and (C) illustrate fusion events and fluorescence decay of IMF, SSM and PN mitochondria under the influence of different treatment groups after HR. The fluorescence decay represents the total reduction of fluorescence intensity from the time of photoactivation to 16 minutes post-photoactivation. $$ denote P<0.01 vs. H.R. + Veh. CT. *, ** and *** denote P<0.05, P<0.01 and P<0.001 vs. Normoxic Time CT, respectively (N=5 for Normoxic Time and Veh. CT and N=4 for Hydralazine).

165 6.3.4 Hydralazine Protects Against HR Induced Cell Death

Subjecting isolated cardiomyocytes to 30 minutes of hypoxia and 15 minutes of reperfusion showed that cells treated with Veh. CT. had significantly higher cell death versus normoxic time CT. (Mean ± S.E. normoxic time CT.: 17.93 ± 2.44 Vs.

34.10 ± 1.51, overall ANOVA P=0.0012) and hydralazine (Mean ± S.E. hydralazine:

24.66 ± 2.77 Vs. 34.10 ± 1.51, overall ANOVA P=0.0012) (See Figure 6-15). In addition, no significant difference was present between hydralazine and normoxic time

CT. groups. Hence, these results affirmed that hydralazine rescues isolated cardiomyocytes from HR induced cell death.

Figure 6-15 Cell Death Assessment for Different Treatment Groups Using PI Staining. The diagram on the left represents the mean percentage of cells with PI and the images on the right panel represent the extent of cell death for different treatment groups.

$ and *** denote P<0.05 and P<0.001 vs. H.R. + Veh. CT. and Normoxic Time CT., respectively (N=5).

166 6.3.5 Ex-vivo Langendorff Hearts Undergoing IRI are Protected by the Action of

Hydralazine

Having seen the protective effect of hydralazine in the setting of mitochondrial dynamics and cell death we then sought to address whether hydralazine has any therapeutic effects on the intact heart. To answer this question, we subjected ex-vivo

Langendorff hearts to 35 minutes of ischaemia and 60 minutes of reperfusion in the presence or absence of hydralazine. As shown in Figure 6-16, there was a clear reduction of infarct size in hearts treated with hydralazine versus the Veh. CT. (Mean

± S.E hydralazine: 29.56% ± 6.53% Vs. 54.09% ± 4.90%, P=0.0083). Taken together, these data showed that hydralazine protects against the detrimental effects of IRI.

Figure 6-16 Infarct Size in Hearts Treated with Either Veh. CT or Hydralazine. The right panel depicts infarct size as a percentage of LV for individual hearts (n=9) and the representative images from each treatment are shown on the left panel.

** denote P<0.01 vs. Veh. CT. (N=9).

167 6.3.6 Hydralazine Targeting of Drp1 and its Effect on Drp1 GTPase activity

To identify the potential mechanism of action of hydralazine we showed via computer simulation in the previous chapter that hydralazine binds to the GTPase domain of Drp1. To assess whether there is a “real” binding of the drug to the Drp1, we performed the SPR assay to detect the dosage and extent of binding of hydralazine to the Drp1 protein. Overall, hydrazine hydrochloride bound in a clear 1:1 kinetic manner with clear on and off rates to the Drp1 protein. The analysis showed a good average deviation from the fitted model and indicated that hydrazine hydrochloride binds to Drp1 with a calculated KD of 8.64 ± 0.92 µM (See Table 6.2 and the example of SPR plot from Flow Cell No.4 in Figure 6-17). Thus, hydralazine truly binds to

Drp1 and can potentially induce its protective effects via modulation of Drp1 function.

Figure 6-17 Example of Flow Cell No.4 SPR Binding Plot. The plot illustrates the reference subtracted curves of different hydralazine concentration in assorted colours and their theoretical curves that are shown in black. Black stars indicate the position of 8.4µM concentration on the plot.

168 Table 6-2 Summary of Hydralazine Binding Kinetics to the Drp1 Protein.

ka (1/Ms) kd (1/s) KD (µM) Rmax (RU) Chi² (RU²)

Flow Cell 4663 0.037 7.99 11.77 0.273 No.4

Flow Cell 3104 0.028 9.29 6.809 0.133 No.2

We next sought to explore the exact modulatory effect of Hydralazine on the GTPase activity of Drp1. As shown in Figure 6-18, incubation of Drp1 with hydralazine significantly decreases its GTPase activity in a dose-dependent manner (Mean ± S.E.:

Veh. CT (0µM): 1 ± 0.05 Vs. 5 µM: 0.77 ± 0.01, 10 µM: 0.60 ± 0.05 and 50 µM: 0.43

± 0.04, ANOVA P < 0.0001). Collectively, the results from here and previous sections show that hydralazine induces its inhibitory effect on mitochondrial fission by binding to the GTPase domain of the Drp1 protein and reducing its activity.

1.2

1

0.8 * *** 0.6 *** 0.4

GTPase activity of Drp1 GTPase activity 0.2 (Fold change alone) Drp1 vs. (Fold change 0 0 5 10 50 Hydralazine hydrochloride (µM)

Figure 6-18 GTPase Activity of Drp1 in the Presence of Different Dosages of Hydralazine. This graph depicts the serial reduction of GTPase activity of Drp1 protein by the increase in the level of Hydralazine (N=3). * and *** denote p<0.05 and p<0.001, respectively.

169 Discussion

This section of the project studied the involvement of mitochondrial dynamics in the induction of hydralazine’s cardioprotective effects following IRI. Here, we initially determined the appropriate dosage of hydralazine and showed that the treatment of HeLa cells with 1µM hydralazine prior to H2O2 insult can prevent ROS- induced fragmentation. We then documented the differences between fusion dynamics of individual subpopulations of mitochondria in adult cardiac myocytes. The fusion events, as well as mitochondrial morphology, were altered upon the induction of HR and these events were rescued by the action of hydralazine. We additionally showed that hydralazine treatment induced protection against cell death and simulated IRI in ex-vivo Langendorff hearts. Finally, we showed that the protective effects of hydralazine may be due to its direct interaction with the Drp1 protein.

Mitochondrial morphology is sensitive to a variety of pathological stimuli such as ROS which is known to participate in mitochondrial fission (237; 354). The process of IRI is known to trigger ROS burst which then causes a reduction of mitochondrial membrane potential followed by mitochondrial fragmentation and subsequent MPTP opening (355). Hydralazine is a potent ROS scavenger and has been shown to reduce superoxide formation (351; 356). Here we found that hydralazine can prevent H2O2 and HR induced mitochondrial fragmentation and membrane potential depolarisation.

These results are consistent with a previous report where they found that the usage of

1µM hydralazine was enough to suppress superoxide formation in isolated adult cardiomyocytes (349). However, here we did not address whether hydralazine preservation of mitochondrial morphology and membrane potential were due to its scavenging effect on H2O2; inhibitory impact on Drp1 GTPase activity or even both scenarios. Hydralazine-mediated inhibition of fragmentation can be linked to its

170 modulatory influence on Drp1 activity. In this chapter and the previous chapter, we showed that the hydralazine binds and decreases the function of the GTPase domain of Drp1, which is known to increase the Drp1 GTPase activity and ultimately facilitate fission (357). This effect of hydralazine is somewhat different from, Mdivi-1, which is known to have little effect on Drp1 GTP hydrolysis function (324). While our study incorporated the GTPase domain of Drp1, we did not cover the other three domains of

Drp1 including VD, middle and GED domain which are suggested to be involved in

Drp1 mitochondrial translocation, self-assembly and retention (358–360). In addition, computer simulation and SPR are cell-free based assays and hence the exact influence of hydralazine on Drp1 binding requires further in-vitro assessment. Lastly, whether the preservation of mitochondrial shape was solely due to the inhibition of fission or it also involved the modulation of fusion proteins remains open for future investigations.

In this report, we additionally looked at the fusion events in adult cardiomyocytes. We observed higher fusion events in IMF mitochondria in comparison to their SSM and PN counterpart. Mitochondrial fusion events are known to be dependent on Ca2+ oscillation and hence it is plausible to hypothesise that Ca2+ handling drives the differences in their fusion dynamics (89; 173–175). Besides, we observed a significant difference in terms of fusion events of IMF mitochondria in comparison to PN mitochondria which are not consistent with a previous report by

Huang et al. (2013) (97). These inconsistencies can be primarily due to the duration they spent for imaging which was 2 hours after photo-activation in comparison to our

16 minutes period. Apart from this difference, the use of cardiomyocytes from different species, the use of viral transfection and the culture incubation period of cardiomyocytes (60-72 hours in comparison to our 24-hours duration) may have also

171 contributed to the disparate findings (97). Furthermore, we observed the preservation of mitochondrial fusion events only in IMF mitochondrial population of adult cardiomyocytes following HR. This mechanism can be related the biochemical properties of different mitochondrial subtypes, and specifically, their calcium regulation (173–175). Hydralazine is known to augment intracellular calcium amplitude and facilitate increased contraction (349) but whether these effects manipulate the fusion dynamics of different subpopulations of mitochondria to elicit cardioprotection remains unclear.

Hydralazine-induced preservation of mitochondrial dynamics was associated with a protective effect against cell death in HR and ex-vivo IRI model. In parallel to these findings, the work by Yang et al. (2011) showed that the in-vivo acute administration of hydralazine can reduce infarct size which was coupled to increased heart rate and mean arterial pressure upon reperfusion. However, the mean percentage of infarct size in their study was 43.3%±2.5% in comparison to our 29.6% ± 6.5%

(333) which can be explained by the differences in our reperfusion times (180 minutes versus our 60 minutes). Chronic treatment of L-NAME-treated spontaneously hypertensive rats (SHR-LNAME) with hydralazine for a period of 3 weeks has been also shown to improve the left ventricular developed pressure and left ventricular end- diastolic pressure following 40 minutes of ischaemia and 30 minutes of reperfusion in comparison to SHR-LNAME rats which did not receive hydralazine (361). However, none of these reports had documented any involvement of mitochondrial dynamics in the observed hydralazine-related cardioprotective effects.

Apart from mitochondrial dynamics, hydralazine is also likely to stabilise

HIF1-α and enhance CGMP expression which are both known to prevent the induction of MPTP (345–351). However, we did not assess whether the cardioprotective effect

172 of hydralazine incorporates the signalling cascades of risk pathway including ERK and

AKT and the subsequent inhibition of MPTP. Hence, the involvement of these aspects in hydralazine-induced cardioprotection is yet to be examined.

Conclusion

In conclusion, mitochondrial dynamics including the changes of mitochondrial morphology and inter-mitochondrial communication in the form of fusion dynamics are of high significance to the adult cardiomyocytes and are altered under pathological

IRI conditions. Hydralazine prevents the detrimental effects of IRI by preserving the mitochondrial dynamics thereby affirming the latter as a therapeutic target for cardioprotection.

173 Chapter 7. Summary and Future Work

The current understanding of the role of mitochondrial dynamics in the homeostasis of adult cardiomyocytes is mainly derived from the study of genetically modified animals (16). This is due to the presence of challenges which have restricted the direct assessment of mitochondrial dynamics in adult cardiomyocytes in vivo. In terms of the morphological evaluation of cardiac mitochondria, the size of adult cardiomyocytes and their relative thick diameter increases the out-of-focus light thereby limiting the axial resolution. In addition, the dense population of mitochondria inside cardiac cells can further limit the axial and lateral resolution which together restrict the assessment of individual mitochondria (272; 274). These aspects ultimately make light microscopy a less favourable technique to study the mitochondrial dynamics in the setting of mitochondrial morphology. As a result, quantification of mitochondrial shape in adult cardiomyocytes is often performed using electron microscopy as this offers a higher resolution in comparison to light microscopy.

Despite offering higher resolution, the electron microscopy evaluation of mitochondrial shape in adult cardiomyocytes is often not detailed, is limited to a single morphometric parameter, and suffers from under-sampling (199; 201; 205; 207). Here we described several 2D and 3D shape descriptors for different mitochondrial subpopulations and showed that they are altered in the presence of pathological conditions. The functional implications induced by changes in these morphological parameters are known to occur in different pathologies (92; 170; 362). However, more work needs to be done to ascertain the exact influence of these shape alterations on the function of the different mitochondrial subpopulations under pathological conditions.

Overall, since the described parameters are unique and independent from each other, they can be used for better and more comprehensive quantification of future 2D and

174 3D evaluation of mitochondrial morphology in adult cardiomyocytes. Moreover, the under-sampling issue associated with quantification of electron microscopy images is often related to manual segmentation and the restricted field of view. As such, the implementation of automatic algorithms capable of detecting mitochondrial shape in all dimensions and the usage of new cutting-edge techniques such as Multi SEM that increase both the speed and the total field of view may drastically improve the quantification accuracy and enhance the extent of data acquisition from individual samples (363; 364).

In addition to morphology, the evaluation of inter-mitochondrial communication in adult cardiomyocytes has been limited due to the lack of probes that can monitor mitochondrial interaction. Using mice expressing Dendra2 fluorescence protein, we have revealed the occurrence of inter-mitochondrial communication in different subpopulations of adult cardiac mitochondria under normal or stress conditions caused by HR. Our results confirmed the results from earlier studies on

H9C2 cardiomyoblast cells (92). Two main conclusions can be drawn from this section of the thesis: first, inter-mitochondrial communication is of high significance to the health of adult cardiomyocytes; and second, it is the morphology that drives the inter- mitochondrial communication. It is also important to mention our current data has proven the existence of inter-mitochondrial communication in isolated adult cardiomyocytes and not in the intact heart. Considering that metabolism of isolated cardiomyocytes may differ from those in the intact heart (89), future experiments conducted using sophisticated microscopy techniques such as multi-photon microscopy may be used to explore the possible differences of these concepts in the whole heart.

175 The notion of mitochondrial dynamics as a target of cardioprotection in the setting of IRI is mainly recognised with the inhibition of mitochondrial fission as a therapeutic strategy (7; 9). Using a HTS of a small molecule library we identified several drugs capable of potentially causing elongation or inhibiting mitochondrial fragmentation. Our screen was the second of its kind to be performed solely on mitochondrial shape and was based on a single morphometric parameter (135). Our

HTS could benefit from the multi-parametric approach of assessing mitochondrial shape, but the utility of the multi-parametric technique in large screens is yet to be validated (317). In addition, since mitochondrial morphology is linked to mitochondrial membrane potential, incorporation of this parameter could have also improved the hit identification (92; 273; 317). However, probes of mitochondrial membrane potential are mainly designed for live cells and the imaging of live cells using HTS is limited due to lack of hardware which allow simultaneous imaging of multiple wells at the same time (365). The hits identified in our screen were capable of binding to Drp1 and possibly inhibiting its effects. The assessment of one these hits, hydralazine, showed that it induces a very similar pattern of protection to Mdivi-1 and preserves mitochondrial morphology after ROS or HR insults as well as protecting the heart against IRI (7). Intriguingly, we demonstrated that hydralazine induces its effect via preservation of inter-mitochondrial communication which adds another dimension to the current view of mitochondrial dynamics as a target for cardioprotection.

Although hydralazine was shown to be therapeutically beneficial in the setting of IRI, its efficacy and application would require in-vivo studies to delineate its exact pharmacological effects. While this thesis proves that mitochondrial dynamics in the form of shape changes and inter-mitochondrial communication are important to the

176 murine adult heart, the relevance of these notions to the human heart is yet to be addressed.

In summary, the work in this thesis has demonstrated the relevance of mitochondrial dynamics in the adult heart highlighting the processes of mitochondrial fusion and fission as future targets for treating cardiovascular disease.

177 Chapter 8. References

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211 Chapter 9. Appendix

Table 9-1 The HTS Results. Drugs Corresponding to the Plate and Position Number Can be Found in Table 9-10.

Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 2 B08 7.69 16 E09 4.27 10 A08 3.26 1 F10 6.86 10 B06 4.26 15 E03 3.25 3 A10 6.64 3 G10 4.23 3 D11 3.23 2 F02 6.47 10 E07 4.22 1 F06 3.2 3 A07 5.83 16 B05 4.17 2 F10 3.18 2 B11 5.64 1 B08 4.13 15 B05 3.18 3 B08 5.58 2 A10 4.13 16 G02 3.17 1 F05 5.58 2 A05 4.1 3 F05 3.13 1 F08 5.46 3 A08 4.09 3 F02 3.13 3 F06 5.45 2 B07 4.09 16 G07 3.12 3 B05 5.4 3 F10 4.07 1 F03 3.11 3 A11 5.31 6 H06 4.03 6 H10 3.08 2 B06 5.27 2 F06 4 11 E10 3.06 14 B11 5.23 16 F08 4 11 E06 3.02 3 A09 5.16 15 B11 3.99 6 F10 3 2 A08 5.12 5 H07 3.94 1 F02 2.95 3 B10 5.1 1 B10 3.89 14 D11 2.94 2 B03 5.06 3 F09 3.88 5 E05 2.92 1 B11 5 12 F11 3.86 11 B03 2.92 3 E10 4.97 16 B07 3.85 2 F08 2.92 5 A10 4.93 1 B06 3.75 13 F04 2.9 3 B11 4.82 1 B09 3.74 5 A09 2.85 15 F03 4.78 5 B06 3.69 15 F06 2.84 16 G08 4.69 15 F11 3.69 5 E06 2.84 2 E10 4.67 3 E08 3.66 5 D05 2.84 3 B06 4.67 2 A03 3.6 8 G09 2.82 3 B09 4.66 1 A10 3.6 13 A02 2.81 10 E02 4.6 5 H06 3.58 7 A09 2.81 3 A04 4.58 5 H10 3.58 15 H04 2.81 15 F10 4.57 9 G09 3.54 1 B07 2.81 3 B04 4.56 10 E08 3.52 2 E06 2.77 2 B09 4.54 15 F04 3.52 2 C10 2.75 5 D08 4.52 2 F03 3.51 16 E02 2.74 5 D10 4.51 16 A03 3.49 8 D11 2.74 5 H11 4.49 15 A11 3.45 12 E04 2.74 12 F03 4.47 15 G11 3.44 2 F04 2.74 15 B08 4.46 16 G10 3.42 15 H06 2.74 1 E11 4.45 7 H06 3.38 16 A08 2.72 16 F05 4.44 3 B07 3.34 1 B02 2.72 5 G10 4.36 2 A04 3.34 10 G06 2.72 2 B05 4.33 10 A09 3.34 2 D07 2.68 2 B10 4.32 1 E09 3.33 2 D09 2.68 11 G09 4.31 2 B04 3.3 15 G06 2.66 1 F07 4.28 3 F03 3.3 16 G03 2.65 12 G09 4.28 1 F09 3.28 16 A11 2.65

212 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 1 E04 2.65 7 H07 2.15 15 E09 1.79 1 E10 2.65 4 A08 2.13 16 D08 1.78 15 F09 2.64 16 E11 2.13 5 G02 1.78 16 A06 2.64 10 G07 2.13 4 E11 1.77 5 F03 2.62 15 D03 2.13 12 D11 1.77 16 G04 2.62 16 G11 2.06 3 A05 1.77 12 G07 2.61 9 C09 2.06 3 E11 1.76 11 F05 2.59 2 A06 2.04 10 E05 1.75 2 G10 2.55 1 D11 2.04 3 H11 1.73 2 D10 2.53 1 H06 2.04 13 B07 1.72 16 A04 2.47 7 G06 2.03 16 B08 1.72 16 E10 2.47 15 E05 2.03 11 F02 1.72 15 E11 2.46 14 H09 2.02 1 B05 1.71 5 H09 2.44 3 E05 2.01 6 D03 1.71 3 B02 2.41 13 B04 2.01 5 A11 1.71 9 H06 2.41 4 D05 2 5 B08 1.71 2 C11 2.38 15 B03 2 4 G05 1.7 15 H02 2.37 15 G07 2 1 B03 1.7 15 F02 2.36 5 E07 1.99 3 B03 1.7 5 D09 2.35 5 G06 1.99 15 E04 1.7 5 H08 2.35 16 B02 1.99 12 A08 1.7 6 E05 2.34 15 B02 1.98 6 D06 1.69 16 B03 2.31 16 E06 1.98 2 F05 1.69 3 C10 2.31 9 G07 1.98 11 F09 1.69 5 H02 2.29 1 D08 1.97 3 E06 1.68 1 A08 2.28 13 B06 1.94 16 G09 1.67 5 C06 2.28 5 G07 1.93 14 B08 1.67 6 G07 2.28 5 E10 1.92 8 D09 1.66 1 F04 2.28 16 B04 1.92 4 H10 1.66 16 E08 2.27 16 B11 1.92 3 C11 1.66 1 A11 2.26 16 F07 1.92 3 G05 1.66 14 H11 2.26 6 D09 1.91 6 D04 1.64 3 D10 2.23 3 G09 1.9 7 B08 1.63 5 G11 2.22 16 B09 1.89 5 E11 1.63 5 H05 2.21 16 D09 1.88 5 F08 1.63 5 D11 2.21 16 F10 1.88 6 E04 1.63 5 D06 2.21 11 E07 1.88 4 G06 1.62 13 B02 2.2 3 A06 1.86 4 A11 1.62 1 A05 2.17 5 F11 1.85 4 D11 1.62 1 F11 2.17 5 B09 1.84 4 D07 1.61 14 E05 2.17 16 B06 1.83 4 E05 1.6 5 E09 2.16 8 C11 1.83 12 F09 1.59 3 F08 2.16 9 B05 1.82 4 G11 1.58 15 B07 2.15 2 E08 1.81 13 A03 1.58 6 D10 2.15 15 A07 1.81 8 A11 1.58

213 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 8 G07 1.57 5 H04 1.35 1 E03 1.05 8 D08 1.56 12 G10 1.34 3 A03 1.03 1 A09 1.56 1 A04 1.34 1 D07 1.02 2 F09 1.56 6 F08 1.33 4 B08 1.02 6 B05 1.56 5 B11 1.33 16 A07 1.01 1 G09 1.55 15 B10 1.32 4 C08 1.01 6 D08 1.54 1 E05 1.3 5 C07 1 6 E06 1.54 4 B02 1.29 14 E08 1 6 E09 1.54 14 E09 1.29 4 H09 1 16 B10 1.53 10 F10 1.28 16 E05 1 15 D11 1.51 14 E10 1.28 3 C09 1 5 C09 1.51 15 E07 1.27 3 D07 1 5 A08 1.5 6 H11 1.27 1 A07 1 6 B09 1.5 6 A05 1.26 8 E06 0.99 8 B09 1.5 6 H05 1.26 5 B10 0.99 8 B05 1.49 3 E09 1.26 9 B04 0.99 6 A11 1.48 6 B10 1.25 9 H07 0.99 12 A09 1.48 6 C08 1.25 9 D04 0.97 12 B06 1.48 8 A07 1.24 6 D05 0.96 1 E08 1.47 1 A06 1.24 2 F07 0.95 6 C07 1.47 12 E11 1.22 13 B03 0.94 2 E04 1.46 12 C06 1.22 1 D10 0.94 13 E07 1.46 8 G08 1.22 12 D09 0.93 16 E07 1.45 7 H05 1.22 16 F09 0.93 5 G09 1.45 8 C02 1.21 13 F09 0.92 15 A03 1.44 8 F07 1.21 3 G07 0.92 2 D08 1.43 16 E03 1.2 6 D11 0.9 2 A07 1.41 15 D09 1.2 10 G09 0.89 5 A05 1.4 11 E03 1.19 2 C03 0.89 5 F05 1.4 14 C10 1.19 10 F06 0.88 5 A04 1.4 3 E07 1.19 6 C05 0.87 6 G02 1.4 9 D09 1.18 4 D08 0.87 13 A07 1.4 6 A07 1.18 15 B06 0.87 14 B07 1.39 8 B06 1.18 12 D06 0.86 15 A08 1.39 3 H10 1.17 10 G11 0.86 1 E07 1.39 3 F11 1.17 14 E07 0.86 15 F08 1.38 7 B09 1.17 14 B10 0.85 3 G08 1.38 2 D04 1.16 3 E02 0.85 14 F07 1.38 2 G11 1.15 12 B07 0.84 2 A09 1.37 13 A08 1.13 12 E08 0.84 15 E08 1.37 2 G06 1.13 5 D04 0.83 13 E03 1.37 2 G03 1.1 16 G05 0.83 1 G07 1.35 1 D04 1.1 8 F11 0.83 5 C10 1.35 9 H09 1.07 6 G05 0.82 5 D07 1.35 5 G03 1.06 9 H02 0.82

214 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 6 C10 0.82 1 B04 0.63 4 A04 0.4 6 G11 0.82 10 G04 0.62 8 D10 0.39 8 A03 0.81 8 G03 0.62 4 G03 0.39 6 G06 0.81 10 F03 0.62 2 G02 0.38 14 D03 0.8 10 F09 0.62 16 H06 0.38 13 F08 0.8 5 D03 0.62 8 E05 0.37 14 B06 0.78 6 E08 0.61 10 B04 0.37 4 G08 0.78 14 B05 0.61 7 G05 0.37 5 C08 0.78 15 H03 0.6 8 H08 0.35 5 G08 0.78 6 E10 0.6 8 D02 0.35 9 B10 0.76 14 E02 0.59 9 A02 0.34 10 G08 0.76 6 H03 0.58 2 E07 0.34 6 G03 0.76 4 A06 0.57 5 C11 0.33 14 A09 0.75 15 B09 0.57 4 A05 0.33 1 C11 0.75 5 B04 0.56 13 A05 0.32 3 D08 0.75 8 D05 0.55 8 B11 0.32 15 E06 0.74 11 A06 0.55 16 F04 0.32 6 A09 0.73 15 B04 0.55 4 F08 0.32 7 E06 0.73 1 H02 0.54 16 C09 0.31 9 A10 0.72 2 C07 0.54 8 G10 0.31 16 F03 0.72 2 C08 0.54 8 H05 0.31 9 D10 0.72 14 G06 0.54 6 F06 0.3 5 F04 0.7 1 C08 0.51 9 C08 0.3 6 D07 0.69 4 D10 0.49 5 F06 0.3 8 H04 0.68 10 B02 0.49 13 F02 0.29 15 E10 0.68 4 D04 0.48 12 B09 0.29 15 F05 0.68 13 A04 0.47 4 A03 0.28 10 F07 0.68 14 E11 0.47 12 F06 0.28 16 D10 0.68 9 A03 0.47 5 H03 0.28 6 C04 0.67 9 B11 0.47 11 G10 0.28 3 G06 0.67 4 H08 0.47 5 A03 0.27 9 G10 0.66 15 H05 0.47 1 D05 0.27 11 B07 0.66 9 C10 0.45 8 B10 0.26 6 C03 0.65 8 H10 0.45 12 G11 0.25 9 D07 0.65 7 B07 0.44 1 C09 0.25 11 A04 0.64 4 H04 0.44 16 H09 0.25 2 E11 0.64 2 A11 0.44 2 A02 0.25 4 B04 0.64 12 B04 0.43 8 G05 0.24 16 F06 0.64 14 A10 0.43 15 A05 0.24 15 A06 0.63 14 H06 0.42 6 A04 0.23 5 B07 0.63 14 A06 0.41 6 A10 0.23 5 E03 0.63 8 A09 0.4 6 F07 0.23 5 F09 0.63 8 F09 0.4 8 C03 0.22 4 C11 0.63 9 B06 0.4 5 C05 0.2 13 E06 0.63 9 G06 0.4 12 B02 0.19

215 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 1 C10 0.19 12 B11 0.03 2 G04 -0.22 7 D02 0.19 4 A07 0.02 3 E04 -0.22 4 A10 0.18 10 A05 0.01 8 F08 -0.22 2 D02 0.18 3 D09 0.01 14 E06 -0.23 13 F07 0.18 9 D06 0 5 A07 -0.23 16 A10 0.18 11 A07 -0.01 5 B05 -0.23 7 F02 0.18 10 H07 -0.02 5 C04 -0.23 8 H09 0.17 4 B03 -0.02 1 E06 -0.23 11 A03 0.16 11 G03 -0.02 14 D02 -0.24 14 C11 0.15 8 E07 -0.02 4 G09 -0.25 12 F08 0.14 7 H08 -0.02 9 G02 -0.25 8 E11 0.14 15 D06 -0.03 12 A03 -0.26 14 F05 0.14 9 C05 -0.04 11 H09 -0.26 12 A05 0.13 15 G05 -0.04 6 G09 -0.26 4 B10 0.13 3 H07 -0.04 2 C09 -0.26 3 H08 0.13 4 B11 -0.04 4 F06 -0.28 6 F04 0.13 2 D03 -0.04 11 F11 -0.29 11 G11 0.13 10 F04 -0.05 4 G02 -0.29 7 F04 0.13 14 B03 -0.05 15 D07 -0.3 7 B06 0.13 2 E03 -0.06 5 C03 -0.3 8 E10 0.12 10 B03 -0.06 5 F10 -0.3 8 H11 0.12 10 G03 -0.07 1 H03 -0.31 11 F10 0.12 15 G10 -0.09 14 A11 -0.31 9 G08 0.12 1 D09 -0.09 5 G05 -0.31 10 H08 0.11 1 A02 -0.1 11 E08 -0.32 14 D10 0.11 15 D05 -0.11 5 C02 -0.33 6 H09 0.11 15 A04 -0.12 9 E10 -0.34 1 G06 0.11 1 D02 -0.13 6 D02 -0.35 12 B08 0.1 15 G03 -0.14 6 H02 -0.35 4 B07 0.1 12 A11 -0.14 6 C06 -0.35 12 H02 0.1 12 A10 -0.15 2 E02 -0.37 16 A09 0.1 16 A05 -0.16 14 F08 -0.37 4 G07 0.09 4 C04 -0.17 15 C10 -0.38 13 A11 0.08 15 A02 -0.18 5 E08 -0.39 15 D08 0.07 9 D03 -0.18 3 G02 -0.4 15 D10 0.06 9 D11 -0.18 3 C08 -0.4 8 B04 0.06 9 C03 -0.2 3 A02 -0.4 1 E02 0.06 6 G08 -0.2 9 H03 -0.4 2 D11 0.05 6 H08 -0.2 3 C07 -0.41 1 H08 0.05 14 F02 -0.21 9 B08 -0.42 14 F10 0.05 1 H10 -0.21 9 B09 -0.42 7 E04 0.05 6 H04 -0.21 9 D08 -0.42 8 F06 0.04 8 A06 -0.21 9 E11 -0.42 7 H03 0.04 8 A08 -0.21 4 E06 -0.42 1 G08 0.03 10 A03 -0.22 3 H06 -0.43

216 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 10 C06 -0.43 6 E03 -0.64 6 A03 -0.84 12 G08 -0.43 3 G11 -0.65 10 B09 -0.84 12 F07 -0.44 1 C06 -0.65 8 F04 -0.84 4 C10 -0.44 8 D07 -0.66 7 H09 -0.84 9 H04 -0.45 3 C04 -0.66 12 G05 -0.84 4 G10 -0.47 5 A06 -0.66 8 A10 -0.84 10 H06 -0.48 7 A08 -0.67 2 E05 -0.84 6 E07 -0.49 12 F02 -0.67 14 G05 -0.84 9 A11 -0.5 11 E09 -0.68 12 C11 -0.84 9 F05 -0.51 12 G02 -0.68 12 A07 -0.85 8 D04 -0.51 8 D06 -0.69 8 C07 -0.86 7 C06 -0.51 8 F10 -0.69 8 E04 -0.86 6 C11 -0.51 10 G10 -0.7 12 F05 -0.86 7 D04 -0.51 11 A11 -0.7 4 A09 -0.86 11 D05 -0.52 8 B08 -0.71 4 F10 -0.86 9 D02 -0.52 2 G08 -0.71 7 C05 -0.86 3 H05 -0.52 12 D03 -0.71 10 G05 -0.87 16 D05 -0.52 12 F04 -0.71 8 C05 -0.88 5 E04 -0.53 6 F09 -0.71 7 F08 -0.9 5 F07 -0.53 1 H11 -0.72 4 F11 -0.9 11 D02 -0.54 2 E09 -0.72 9 H11 -0.91 10 F05 -0.54 3 E03 -0.74 5 A02 -0.91 12 F10 -0.55 8 G02 -0.74 9 C06 -0.91 16 F02 -0.55 14 D05 -0.74 9 A06 -0.91 13 E05 -0.55 4 E09 -0.74 11 E02 -0.92 12 B05 -0.56 12 E02 -0.76 4 G04 -0.93 1 G11 -0.56 10 B05 -0.76 16 G06 -0.93 9 C07 -0.56 13 B05 -0.76 4 H11 -0.93 16 C04 -0.57 13 B08 -0.77 12 H03 -0.93 4 C09 -0.57 13 F05 -0.77 14 E03 -0.94 6 B07 -0.57 9 F06 -0.77 8 C08 -0.94 14 A08 -0.57 11 H02 -0.77 12 H09 -0.95 7 G03 -0.58 7 D06 -0.77 11 D03 -0.95 3 D02 -0.59 8 C09 -0.77 12 E06 -0.95 3 F04 -0.59 6 F05 -0.77 8 C10 -0.96 7 C02 -0.6 11 A02 -0.78 11 F07 -0.96 2 H11 -0.6 8 E08 -0.78 9 B02 -0.97 9 F09 -0.6 7 A03 -0.79 9 F04 -0.97 5 B02 -0.6 8 G04 -0.79 10 D06 -0.98 5 E02 -0.6 6 B06 -0.8 4 B09 -1 10 E06 -0.6 4 D09 -0.81 12 E10 -1 16 D07 -0.61 7 F05 -0.81 1 D06 -1.01 5 D02 -0.62 14 G07 -0.81 4 B05 -1.02 7 C09 -0.62 10 A07 -0.82 6 H07 -1.02 10 B07 -0.63 1 G04 -0.83 8 B07 -1.02

217 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 8 F05 -1.02 2 B02 -1.31 11 E04 -1.62 14 D08 -1.03 12 C09 -1.32 9 D05 -1.62 15 H07 -1.04 1 D03 -1.33 4 H07 -1.62 9 A05 -1.04 9 G11 -1.33 16 A02 -1.63 15 C11 -1.04 7 D07 -1.33 4 F07 -1.63 1 A03 -1.04 4 C05 -1.34 4 H06 -1.63 7 E07 -1.05 16 C08 -1.34 15 G02 -1.63 4 D06 -1.06 8 A02 -1.34 9 C04 -1.63 8 E02 -1.07 14 F06 -1.35 3 G04 -1.64 4 F09 -1.08 7 F07 -1.35 2 C04 -1.65 6 B11 -1.09 8 A05 -1.36 6 B04 -1.66 3 F07 -1.09 6 E11 -1.37 6 B08 -1.66 13 B11 -1.1 15 H09 -1.37 6 G04 -1.66 14 B04 -1.11 7 H04 -1.37 9 E03 -1.66 14 D06 -1.12 10 E09 -1.38 9 F08 -1.68 14 D07 -1.12 12 G04 -1.39 11 B02 -1.68 12 B03 -1.12 4 H05 -1.39 15 C09 -1.68 8 E03 -1.12 14 C08 -1.39 1 C03 -1.69 8 G06 -1.13 14 D04 -1.39 7 E03 -1.7 6 A06 -1.14 4 H03 -1.4 12 E05 -1.7 14 C09 -1.15 11 G04 -1.41 12 A06 -1.7 6 F11 -1.15 16 D06 -1.44 16 C06 -1.71 1 G03 -1.15 4 E03 -1.46 6 B03 -1.72 3 G03 -1.16 7 C04 -1.47 12 A04 -1.72 8 A04 -1.17 4 C07 -1.47 4 E07 -1.72 10 A06 -1.17 4 E08 -1.47 14 B02 -1.73 14 B09 -1.18 15 A09 -1.48 10 F02 -1.74 12 D07 -1.2 4 H02 -1.49 3 D03 -1.74 14 A07 -1.2 6 C02 -1.52 15 C07 -1.74 11 B05 -1.2 10 D07 -1.53 4 D03 -1.74 10 G02 -1.2 5 B03 -1.54 14 C07 -1.76 6 C09 -1.21 4 F04 -1.55 9 E07 -1.76 8 F03 -1.21 4 F05 -1.55 16 F11 -1.77 6 G10 -1.22 12 H10 -1.55 1 H09 -1.77 16 D03 -1.22 16 H03 -1.56 7 E02 -1.77 7 B03 -1.23 15 G04 -1.56 9 B03 -1.77 5 G04 -1.23 2 H03 -1.57 12 E09 -1.79 11 B10 -1.24 14 F03 -1.57 13 D07 -1.8 12 E03 -1.25 14 F04 -1.57 14 A05 -1.8 11 F04 -1.26 10 C08 -1.57 6 E02 -1.81 13 A10 -1.27 11 B04 -1.59 10 D10 -1.81 16 C07 -1.28 7 G04 -1.6 4 E10 -1.81 14 F11 -1.28 15 H08 -1.6 14 A04 -1.81 8 B03 -1.3 1 C07 -1.61 11 D04 -1.82 11 B09 -1.3 13 E09 -1.61 3 D04 -1.82

218 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 13 E08 -1.83 11 E05 -2.13 13 C05 -2.48 9 A04 -1.83 10 D02 -2.14 7 G11 -2.48 13 F06 -1.83 8 H07 -2.14 7 C08 -2.5 9 G03 -1.84 16 C11 -2.16 9 F11 -2.5 13 E04 -1.85 12 E07 -2.17 7 D05 -2.5 1 C04 -1.85 7 G08 -2.18 13 F03 -2.53 1 G05 -1.85 9 F02 -2.2 14 H07 -2.55 10 A02 -1.86 2 G07 -2.2 2 H04 -2.57 10 B10 -1.87 10 B11 -2.21 8 G11 -2.57 1 C05 -1.87 8 E09 -2.21 2 H09 -2.61 1 G10 -1.88 13 A09 -2.22 8 B02 -2.61 11 B11 -1.89 3 C02 -2.23 12 C03 -2.62 3 C03 -1.9 6 A08 -2.25 7 F03 -2.62 9 B07 -1.91 16 D02 -2.25 11 B08 -2.63 12 C05 -1.93 15 G08 -2.27 7 A07 -2.65 9 E04 -1.93 4 C06 -2.29 13 G03 -2.66 9 E09 -1.93 14 F09 -2.29 11 A08 -2.66 2 H07 -1.93 7 F09 -2.29 11 G08 -2.67 4 B06 -1.93 16 H04 -2.29 5 F02 -2.68 7 A06 -1.94 7 B04 -2.32 11 H07 -2.7 6 A02 -1.95 7 D08 -2.33 8 F02 -2.71 6 B02 -1.96 15 G09 -2.34 14 D09 -2.72 3 C06 -1.99 12 A02 -2.34 7 E09 -2.72 7 C03 -2 1 C02 -2.35 11 A09 -2.72 12 G03 -2.02 1 H04 -2.35 14 C04 -2.74 8 D03 -2.02 14 E04 -2.36 4 C02 -2.74 9 E08 -2.03 7 B02 -2.37 4 E04 -2.74 7 G07 -2.04 7 F06 -2.37 4 E02 -2.75 6 F03 -2.04 11 A05 -2.37 13 G07 -2.76 8 C06 -2.05 8 C04 -2.38 10 C02 -2.77 11 F08 -2.05 9 A09 -2.38 15 C06 -2.78 4 A02 -2.05 10 H09 -2.38 11 D09 -2.79 15 A10 -2.07 16 D04 -2.41 7 C10 -2.8 10 A10 -2.07 7 B05 -2.42 11 C02 -2.81 12 C10 -2.08 16 C10 -2.42 9 A08 -2.82 9 G04 -2.08 9 A07 -2.43 9 C02 -2.85 15 C08 -2.09 7 G09 -2.43 7 F10 -2.86 7 D03 -2.09 12 C08 -2.44 7 E10 -2.86 14 A02 -2.09 10 E04 -2.44 15 D04 -2.86 14 G03 -2.1 13 D02 -2.44 7 E05 -2.88 12 C07 -2.1 10 D03 -2.44 13 B10 -2.88 7 C07 -2.12 2 F11 -2.45 10 C09 -2.89 14 A03 -2.12 14 C05 -2.46 3 D06 -2.9 12 D04 -2.12 7 G02 -2.47 4 D02 -2.9 8 H06 -2.12 16 E04 -2.47 4 F02 -2.9

219 Plate No Position Z-score Plate Position Z- Plate Position Z- No No No score No No score 4 F03 -2.91 11 C10 -3.39 10 D08 -4.17 6 F02 -2.91 11 G07 -3.39 16 H05 -4.17 9 H08 -2.92 10 C07 -3.41 11 C05 -4.21 9 E05 -2.92 10 H03 -3.43 3 D05 -4.22 10 A04 -2.92 12 H04 -3.43 11 E11 -4.25 14 C03 -2.92 13 E11 -3.5 13 D04 -4.3 7 A04 -2.95 13 D06 -3.5 12 H11 -4.32 10 E10 -2.97 13 D05 -3.53 13 G09 -4.36 2 C02 -2.97 15 H11 -3.54 14 G04 -4.37 3 H09 -2.99 3 H04 -3.55 2 C06 -4.47 15 C05 -2.99 14 C02 -3.57 13 C07 -4.5 12 D08 -3 14 H05 -3.59 10 H04 -4.51 4 C03 -3.01 7 F11 -3.6 11 H05 -4.57 9 F03 -3.01 11 G02 -3.62 3 H02 -4.57 10 H11 -3.02 7 H02 -3.65 2 H02 -4.58 1 H07 -3.03 13 C03 -3.65 10 C10 -4.59 10 B08 -3.03 15 H10 -3.71 11 D07 -4.65 7 E08 -3.03 7 D10 -3.72 12 H05 -4.76 7 A02 -3.04 10 C05 -3.76 11 A10 -4.78 12 D05 -3.07 11 H11 -3.79 11 C07 -4.81 9 E02 -3.07 12 D02 -3.79 10 E03 -4.85 13 A06 -3.07 11 G06 -3.79 3 H03 -4.89 12 G06 -3.08 3 C05 -3.8 10 D04 -4.9 16 C05 -3.08 15 C04 -3.85 13 E02 -5.02 2 D06 -3.08 15 C03 -3.87 11 C09 -5.07 11 D08 -3.1 13 G11 -3.88 10 C04 -5.07 13 C06 -3.1 7 A05 -3.9 10 F08 -5.1 2 H10 -3.11 15 E02 -3.91 11 D06 -5.1 12 D10 -3.11 11 G05 -3.91 2 H05 -5.14 2 D05 -3.13 11 C03 -3.92 13 C02 -5.14 16 C03 -3.15 7 D09 -3.94 16 C02 -5.24 10 E11 -3.16 16 H08 -3.95 11 D10 -5.25 13 C04 -3.18 15 F07 -3.95 1 G02 -5.25 11 D11 -3.2 14 G02 -3.95 10 A11 -5.31 13 F10 -3.23 2 G05 -3.96 15 D02 -5.39 11 C11 -3.24 2 G09 -3.99 2 C05 -5.42 11 C04 -3.28 13 D03 -4.02 13 C08 -5.44 11 F03 -3.3 11 H06 -4.02 12 B10 -5.44 14 G08 -3.31 14 C06 -4.05 13 G04 -5.46 14 H04 -3.31 10 F11 -4.08 7 C11 -5.55 14 G11 -3.33 13 C09 -4.09 7 D11 -5.56 9 E06 -3.35 11 H04 -4.11 11 F06 -5.7 11 B06 -3.35 10 D09 -4.13 12 H07 -5.73 13 B09 -3.36 10 C03 -4.14 1 H05 -5.73 13 E10 -3.38 11 C06 -4.16 11 H08 -5.77

220 Plate No Position Z-score Plate Position Z-score No No No 11 C08 -5.79 10 D05 -18.88 14 H02 -5.82 2 H06 * 13 H10 -6.01 7 A10 * 12 C02 -6.06 7 A11 * 15 C02 -6.17 7 A12 * 13 C11 -6.17 7 B10 * 12 C04 -6.2 7 B11 * 9 G05 -6.2 7 B12 * 16 D11 -6.29 7 H10 * 10 D11 -6.34 8 G12 * 13 F11 -6.34 8 H02 * 13 H06 -6.36 8 H03 * 11 H03 -6.42 9 H05 * 13 H08 -6.43 9 H10 * 14 H10 -6.44 12 H08 * 13 D08 -6.54 13 G06 * 7 E11 -6.54 13 G08 * 13 G02 -6.56 13 G10 * 7 G10 -6.79 14 G09 * 14 G10 -7.09 15 G12 * 2 H08 -7.1 16 H02 * 14 H08 -7.16 16 H07 * 14 H03 -7.2 16 H10 * 12 H06 -7.32 16 H11 * 13 H03 -7.39 11 H10 -7.49 9 C11 -7.52 10 H10 -7.57 10 H02 -7.66 9 F07 -7.69 13 C10 -7.81 10 H05 -7.83 13 D10 -7.94 13 H02 -8.23 13 D11 -8.33 10 C11 -8.39 13 G05 -8.69 13 H07 -8.84 13 H05 -9.03 13 H09 -9.52 13 D09 -10.59 9 F10 -11.9 13 H04 -12.47 7 H11 -13.15 13 H11 -18.32 * Drugs with empty wells

221 Table 9-2 List of Drugs in Prestwick Chemical Library. For the Z-score of Individual drugs Refer to Table 9-1.

Plate Mol NO / Therapeutic Chemical name weight Therapeutic effect Position class

NO 01A02 Azaguanine-8 152.12 Oncology Antineoplastic 01A03 Allantoin 158.12 Dermatology Antipsoriatic 01A04 Acetazolamide 222.25 Metabolism Anticonvulsant 01A05 Metformin hydrochloride 165.63 Endocrinology Anorectic 01A06 Atracurium besylate 1243.51 Neuromuscular Curarizing 01A07 Isoflupredone acetate 420.48 Endocrinology Anti-inflammatory Amiloride hydrochloride 01A08 302.12 Metabolism Antihypertensive dihydrate 01A09 Amprolium hydrochloride 315.25 Infectiology Anticoccidial 01A10 Hydrochlorothiazide 297.74 Metabolism Antihypertensive 01A11 Sulfaguanidine 214.25 Infectiology Antibacterial 01B02 Meticrane 275.35 Metabolism Antihypertensive 01B03 Benzonatate 603.76 Neuromuscular Antitussive 01B04 Hydroflumethiazide 331.29 Metabolism Antihypertensive 01B05 Sulfacetamide sodic hydrate 254.24 Dermatology Antibacterial 01B06 Heptaminol hydrochloride 181.71 Cardiovascular Analeptic 01B07 Sulfathiazole 255.32 Infectiology Antibacterial Central Nervous 01B08 Levodopa 197.19 Antiparkinsonian System 01B09 Idoxuridine 354.10 Infectiology Antiviral 01B10 Captopril 217.29 Cardiovascular Antihypertensive 01B11 Minoxidil 209.25 Cardiovascular Anti-alopecia 01C02 Sulfaphenazole 314.37 Infectiology Antibacterial 01C03 Panthenol (D) 205.26 Metabolism Anti-alopecia 01C04 Sulfadiazine 250.28 Infectiology Antibacterial 01C05 Norethynodrel 298.43 Endocrinology Contraceptive 01C06 Thiamphenicol 356.23 Infectiology Antibacterial 01C07 Cimetidine 252.34 Gastroenterology Antiulcer 01C08 Doxylamine succinate 388.47 Allergology Anti-anorectic 01C09 Ethambutol dihydrochloride 277.24 Infectiology Antibacterial Central Nervous 01C10 Antipyrine 188.23 Analgesic System 01C11 Antipyrine, 4-hydroxy 204.23 Metabolism 01D02 Chloramphenicol 323.13 Infectiology Antibacterial Central Nervous 01D03 Epirizole 234.26 Analgesic System 01D04 Diprophylline 254.25 Cardiovascular Analeptic 01D05 Triamterene 253.27 Metabolism Antihypertensive 01D06 Dapsone 248.31 Infectiology Antibacterial 01D07 Troleandomycin 813.99 Infectiology Antibacterial 01D08 Pyrimethamine 248.72 Infectiology Antimalarial Hexamethonium dibromide 01D09 398.22 Cardiovascular Antihypertensive dihydrate Central Nervous 01D10 Diflunisal 250.20 Analgesic System 01D11 Niclosamide 327.13 Infectiology Antihelmintic

222 01E02 Procaine hydrochloride 272.78 Neuromuscular Local anaesthetic Erectile dysfunction 01E03 Moxisylyte hydrochoride 315.84 Cardiovascular treatment 01E04 Betazole hydrochloride 184.07 Gastroenterology Diagnostic Central Nervous 01E05 Isoxicam 335.34 Analgesic System Central Nervous 01E06 Naproxen 230.27 Analgesic System 01E07 Naphazoline hydrochloride 246.74 Cardiovascular Nasal Decongestant 01E08 Ticlopidine hydrochloride 300.25 Hematology Anticoagulant 01E09 Dicyclomine hydrochloride 345.96 Gastroenterology Antispastic 01E10 Amyleine hydrochloride 271.79 Neuromuscular Local anaesthetic 01E11 Lidocaine hydrochloride 270.81 Cardiovascular Antiarrhythmic 01F02 Mupirocin 500.64 Infectiology Central Nervous 01F03 Carbamazepine 236.28 Analgesic System Triflupromazine Central Nervous 01F04 388.89 Antiemetic hydrochloride System Central Nervous 01F05 Mefenamic acid 241.29 Analgesic System 01F06 Acetohexamide 324.40 Endocrinology Antidiabetic Central Nervous 01F07 Sulpiride 341.43 Antidepressant System 01F08 Benoxinate hydrochloride 344.89 Neuromuscular Local anaesthetic 01F09 Oxethazaine 467.66 Neuromuscular Local anaesthetic 01F10 Pheniramine maleate 356.43 Allergology Antihistaminic 01F11 Tolazoline hydrochloride 196.68 Cardiovascular Vasodilator 01G02 Morantel tartrate 370.43 Infectiology Antihelmintic Homatropine hydrobromide 01G03 356.26 Diagnostic Antispastic (R,S) 01G04 Nifedipine 346.34 Cardiovascular Antianginal Chlorpromazine 01G05 355.33 Cardiovascular Antiemetic hydrochloride Diphenhydramine 01G06 291.82 Allergology Antiemetic hydrochloride Central Nervous 01G07 Minaprine dihydrochloride 371.31 Anti-Alzheimer System 01G08 Miconazole 416.14 Infectiology Antifungal 01G09 Isoxsuprine hydrochloride 337.85 Cardiovascular Vasodilator 01G10 Acebutolol hydrochloride 372.90 Cardiovascular Antianginal 01G11 Tolnaftate 307.42 Infectiology Antifungal 01H02 Todralazine hydrochloride 268.70 Cardiovascular Antihypertensive Central Nervous 01H03 Imipramine hydrochloride 316.88 Antidepressant System Central Nervous 01H04 Sulindac 356.42 Analgesic System Central Nervous 01H05 Amitryptiline hydrochloride 313.87 Antidepressant System 01H06 Adiphenine hydrochloride 347.89 Neuromuscular Antispastic 01H07 Dibucaine 343.47 Neuromuscular Local anaesthetic 01H08 Prednisone 358.44 Dermatology Anti-inflammatory Central Nervous 01H09 Thioridazine hydrochloride 407.04 Antipsychotic System 01H10 Diphemanil methylsulfate 389.52 Gastroenterology Antispastic

223 Trimethobenzamide Central Nervous 01H11 424.93 Antiemetic hydrochloride System 02A02 Metronidazole 171.16 Infectiology Antiamebic 02A03 Fulvestrant 606.79 Endocrinology Antineoplastic 02A04 Edrophonium chloride 201.70 Diagnostic Anti-fatigue 02A05 Moroxidine hydrochloride 207.66 Infectiology Antiviral Central Nervous 02A06 Baclofen (R,S) 213.67 Antispastic System 02A07 Acyclovir 225.21 Metabolism Antiviral 02A08 Diazoxide 230.67 Cardiovascular Antidiuretic Central Nervous 02A09 Amidopyrine 231.30 Analgesic System 02A10 Busulfan 246.30 Oncology Antineoplastic 02A11 Pindolol 248.33 Cardiovascular Antianginal 02B02 Khellin 260.25 Cardiovascular Antispastic Zimelidine dihydrochloride Central Nervous 02B03 408.17 Antidepressant monohydrate System Central Nervous 02B04 Azacyclonol 267.37 Antipsychotic System 02B05 Azathioprine 277.27 Oncology Antineoplastic 02B06 Lynestrenol 284.45 Endocrinology Contraceptive Central Nervous 02B07 Guanabenz acetate 291.14 Antihypertensive System 02B08 Disulfiram 296.54 Metabolism Antabuse effect Central Nervous 02B09 Acetylsalicylsalicylic acid 300.27 Analgesic System 02B10 Mianserine hydrochloride 300.83 Cardiovascular Antidepressant 02B11 Nocodazole 301.33 Oncology Antineoplastic R(-) Apomorphine Central Nervous 02C02 625.60 Antiparkinsonian hydrochloride hemihydrate System Central Nervous 02C03 Amoxapine 313.79 Antidepressant System Cyproheptadine 02C04 323.87 Allergology Antihistaminic hydrochloride 02C05 Famotidine 337.45 Gastroenterology Antiulcer 02C06 Danazol 337.47 Endocrinology Anabolic 02C07 Nicorandil 211.18 Cardiovascular Antianginal 02C08 Pioglitazone 356.45 Endocrinology Central Nervous 02C09 Nomifensine maleate 354.41 Antidepressant System Central Nervous 02C10 Dizocilpine maleate 337.38 Anticonvulsant System 02C11 Oxandrolone 306.45 Endocrinology Central Nervous 02D02 Naloxone hydrochloride 363.84 Opioate antidote System 02D03 Metolazone 365.84 Cardiovascular Antihypertensive Ciprofloxacin hydrochloride 02D04 385.83 Infectiology Antibacterial monohydrate 02D05 Ampicillin trihydrate 403.46 Infectiology Antibacterial Central Nervous 02D06 Haloperidol 375.87 Antiemetic System Naltrexone hydrochloride Central Nervous 02D07 413.90 Analgesic dihydrate System 02D08 Chlorpheniramine maleate 390.87 Allergology Antihistaminic

224 Central Nervous 02D09 Nalbuphine hydrochloride 393.91 Analgesic System 02D10 Picotamide monohydrate 394.43 Hematology Anticoagulant 02D11 Triamcinolone 394.44 Endocrinology Anti-inflammatory Central Nervous 02E02 Bromocryptine mesylate 750.72 Antiparkinsonian System Central Nervous 02E03 Amfepramone hydrochloride 241.76 System 02E04 Dehydrocholic acid 402.54 Gastroenterology Choleretic 02E05 Tioconazole 387.72 Infectiology Antifungal Central Nervous 02E06 Perphenazine 403.98 Antiemetic System 02E07 Mefloquine hydrochloride 414.78 Infectiology Antimalarial 02E08 Isoconazole 416.14 Infectiology Antibacterial 02E09 Spironolactone 416.58 Endocrinology Diuretic 02E10 Pirenzepine dihydrochloride 424.33 Gastroenterology Antiulcer 02E11 Dexamethasone acetate 434.51 Endocrinology Anti-inflammatory 02F02 Glipizide 445.54 Endocrinology Antidiabetic 02F03 Loxapine succinate 445.91 Central Nervous Antipsychotic System 02F04 Hydroxyzine 447.84 Allergology Antiemetic dihydrochloride 02F05 Diltiazem hydrochloride 450.99 Cardiovascular Antianginal 02F06 Methotrexate 454.45 Oncology Antineoplastic 02F07 Astemizole 458.58 Allergology Antihistaminic 02F08 Clindamycin hydrochloride 461.45 Infectiology Antibacterial 02F09 Terfenadine 471.69 Allergology Antihistaminic 02F10 Cefotaxime sodium salt 477.45 Infectiology Antibacterial 02F11 Tetracycline hydrochloride 480.91 Infectiology Antibacterial 02G02 Verapamil hydrochloride 491.08 Cardiovascular Antihypertensive 02G03 Dipyridamole 504.64 Cardiovascular Anticoagulant 02G04 Chlorhexidine 505.46 Infectiology Antibacterial 02G05 Loperamide hydrochloride 513.51 Gastroenterology Antidiarrheal 02G06 Chlortetracycline 515.35 Infectiology Antiamebic hydrochloride 02G07 Tamoxifen citrate 563.65 Endocrinology Antineoplastic 02G08 Nicergoline 484.40 Cardiovascular Anti-ischemic 02G09 Canrenoic acid potassium 396.58 Endocrinology Antihypertensive salt 02G10 Thioproperazine dimesylate 638.85 Central Nervous Antiemetic System 02G11 Dihydroergotamine tartrate 1317.48 Central Nervous Antimigraine System 02H02 Erythromycin 733.95 Infectiology Antibacterial 02H03 Chloroxine 214.05 Dermatology 02H04 Didanosine 236.23 Infectiology Antiviral 02H05 Josamycin 828.02 Infectiology Antibacterial 02H06 Paclitaxel 853.93 Oncology Antineoplastic 02H07 Ivermectin 875.12 Infectiology Antihelmintic 02H08 Gallamine triethiodide 891.54 Neuromuscular Muscle relaxant 02H09 Neomycin sulfate 712.73 Infectiology Antibacterial 02H10 Dihydrostreptomycin sulfate 1461.43 Infectiology Antibacterial 02H11 Gentamicine sulfate 1488.81 Infectiology Antibacterial 03A02 Isoniazid 137.14 Infectiology Antibacterial

225 03A03 Pentylenetetrazole 138.17 Central Nervous Analeptic System 03A04 Chlorzoxazone 169.57 Central Nervous Anticonvulsant System 03A05 Ornidazole 219.63 Infectiology Antibacterial 03A06 Ethosuximide 141.17 Central Nervous Anticonvulsant System 03A07 Mafenide hydrochloride 222.69 Infectiology Antibacterial 03A08 Riluzole hydrochloride 270.66 Central Nervous Antispastic System 03A09 Nitrofurantoin 238.16 Infectiology Antibacterial 03A10 Hydralazine hydrochloride 196.64 Cardiovascular Antihypertensive 03A11 Phenelzine sulfate 234.28 Central Nervous Antidepressant System 03B02 Tranexamic acid 157.21 Hematology Hemostatic 03B03 Etofylline 224.22 Cardiovascular Antispastic 03B04 Tranylcypromine 169.66 Central Nervous Antidepressant hydrochloride System 03B05 Alverine citrate salt 473.57 Neuromuscular Antispastic 03B06 Aceclofenac 354.19 Central Nervous Analgesic System 03B07 Iproniazide phosphate 277.22 Cardiovascular Antidepressant 03B08 Sulfamethoxazole 253.28 Infectiology Antibacterial 03B09 Mephenesin 182.22 Central Nervous Anticonvulsant System 03B10 Phenformin hydrochloride 241.73 Endocrinology Antidiabetic 03B11 Flutamide 276.22 Oncology Antineoplastic 03C02 Ampyrone 203.25 Central Nervous Analgesic System 03C03 Levamisole hydrochloride 240.76 Immunology Antihelmintic 03C04 Pargyline hydrochloride 195.69 Cardiovascular Antidepressant 03C05 Methocarbamol 241.25 Central Nervous Analgesic System 03C06 Aztreonam 435.44 Infectiology Antibacterial 03C07 Cloxacillin sodium salt 457.87 Infectiology Antibacterial 03C08 Catharanthine 336.44 Oncology Antineoplastic 03C09 Pentolinium bitartrate 538.60 Cardiovascular Antihypertensive 03C10 Aminopurine, 6-benzyl 225.25 Endocrinology 03C11 Tolbutamide 270.35 Endocrinology Antidiabetic 03D02 Midodrine hydrochloride 290.75 Cardiovascular Antihypertensive 03D03 Thalidomide 258.24 Central Nervous Hypnotic System 03D04 Oxolinic acid 261.24 Metabolism Antibacterial 03D05 Nimesulide 308.31 Metabolism Anti-inflammatory 03D06 Asenapine maleate 401.85 Central Nervous Antipsychotic System 03D07 Pentoxifylline 278.31 Cardiovascular Bronchodilator 03D08 Metaraminol bitartrate 467.39 Cardiovascular Antihypotensive 03D09 Salbutamol 239.32 Neuromuscular Bronchodilator 03D10 Prilocaine hydrochloride 256.78 Neuromuscular Local anaesthetic 03D11 Camptothecine (S,+) 348.36 Oncology Antineoplastic 03E02 Ranitidine hydrochloride 350.87 Gastroenterology Antiulcer 03E03 Tiratricol, 3,3',5- 621.94 Endocrinology Antihypothyroid triiodothyroacetic acid

226 03E04 Flufenamic acid 281.24 Central Nervous Analgesic System 03E05 Flumequine 261.26 Infectiology Antibacterial 03E06 Tolfenamic acid 261.71 Central Nervous Analgesic System 03E07 Meclofenamic acid sodium 336.15 Central Nervous Anti-inflammatory salt monohydrate System 03E08 Tibolone 312.46 Endocrinology 03E09 Trimethoprim 290.32 Infectiology Antibacterial 03E10 Metoclopramide 336.26 Central Nervous Antiemetic monohydrochloride System 03E11 Fenbendazole 299.35 Infectiology Antihelmintic 03F02 Piroxicam 331.35 Central Nervous Analgesic System 03F03 Pyrantel tartrate 356.40 Infectiology Antihelmintic 03F04 Fenspiride hydrochloride 296.80 Respiratory Antitussive 03F05 Gemfibrozil 250.34 Metabolism Hypocholesterolemic 03F06 Mefexamide hydrochloride 316.83 Central Nervous CNS Stimulant System 03F07 Tiapride hydrochloride 364.89 Central Nervous Antiemetic System 03F08 Mebendazole 295.30 Infectiology Antihelmintic 03F09 Fenbufen 254.29 Central Nervous Analgesic System 03F10 Ketoprofen 254.29 Central Nervous Analgesic System 03F11 Indapamide 365.84 Metabolism Antihypertensive 03G02 Norfloxacin 319.34 Infectiology Antibacterial 03G03 Antimycin A 548.64 Infectiology Antibacterial 03G04 Xylometazoline 280.84 Cardiovascular Nasal Decongestant hydrochloride 03G05 Oxymetazoline 296.84 Respiratory Nasal Decongestant hydrochloride 03G06 Nifenazone 308.34 Central Nervous Analgesic System 03G07 Griseofulvin 352.77 Infectiology Antifungal 03G08 Clemizole hydrochloride 362.31 Allergology Antibacterial 03G09 Tropicamide 284.36 Neuromuscular Mydriatic 03G10 Nefopam hydrochloride 289.81 Central Nervous Analgesic System 03G11 Phentolamine hydrochloride 317.82 Cardiovascular Antihypertensive 03H02 Etodolac 287.36 Central Nervous Analgesic System 03H03 Scopolamin-N-oxide 400.27 Neuromuscular Antispastic hydrobromide 03H04 Hyoscyamine (L) 289.38 Central Nervous Antiemetic System 03H05 Chlorphensin carbamate 245.66 Central Nervous Muscle relaxant System 03H06 Carmofur 257.27 Metabolism Antineoplastic 03H07 Dilazep dihydrochloride 677.63 Cardiovascular Antiplatelet 03H08 Ofloxacin 361.38 Infectiology Antibacterial 03H09 Lomefloxacin hydrochloride 387.82 Infectiology Antibacterial 03H10 Orphenadrine hydrochloride 305.85 Allergology Antihistaminic 03H11 Proglumide 334.42 Gastroenterology Antiulcer

227 04A02 Mexiletine hydrochloride 215.73 Cardiovascular Antiarrhythmic 04A03 Flavoxate hydrochloride 427.93 Metabolism Antispastic 04A04 Bufexamac 223.27 Central Nervous Analgesic System 04A05 Glutethimide, para-amino 232.28 Oncology Antineoplastic 04A06 Dropropizine (R,S) 236.32 Respiratory Antitussive 04A07 Pinacidil 245.33 Cardiovascular Antihypertensive 04A08 Albendazole 265.34 Metabolism Antihelmintic 04A09 Clonidine hydrochloride 266.56 Cardiovascular Analgesic 04A10 Bupropion hydrochloride 276.21 Central Nervous Antidepressant System 04A11 Alprenolol hydrochloride 285.82 Cardiovascular Antianginal 04B02 Chlorothiazide 295.72 Metabolism Antihypertensive 04B03 Diphenidol hydrochloride 345.92 Central Nervous Antiemetic System 04B04 Norethindrone 298.43 Endocrinology Contraceptive 04B05 Nortriptyline hydrochloride 299.85 Central Nervous Antidepressant System 04B06 Niflumic acid 282.22 Central Nervous Analgesic System 04B07 Isotretinoin 300.44 Dermatology Keratolytic 04B08 Retinoic acid 300.44 Dermatology Keratolytic 04B09 Antazoline hydrochloride 301.82 Allergology Antihistaminic 04B10 Ethacrynic acid 303.14 Metabolism Diuretic 04B11 Praziquantel 312.42 Infectiology Antihelmintic 04C02 Ethisterone 312.46 Endocrinology Contraceptive 04C03 Triprolidine hydrochloride 314.86 Allergology Antihistaminic 04C04 Doxepin hydrochloride 315.85 Allergology Anticonvulsant 04C05 Dyclonine hydrochloride 325.88 Neuromuscular Local anaesthetic 04C06 Dimenhydrinate 469.98 Allergology Antiemetic 04C07 Disopyramide 339.48 Cardiovascular Antiarrhythmic 04C08 Clotrimazole 344.85 Infectiology Antibacterial 04C09 Vinpocetine 350.46 Cardiovascular CNS Stimulant 04C10 Clomipramine hydrochloride 351.32 Central Nervous Antidepressant System 04C11 Fendiline hydrochloride 351.92 Cardiovascular Antianginal 04D02 Vincamine 354.45 Central Nervous CNS Stimulant System 04D03 Indomethacin 357.80 Central Nervous Analgesic System 04D04 Cortisone 360.45 Endocrinology Anti-inflammatory 04D05 Prednisolone 360.45 Endocrinology Anti-inflammatory 04D06 Fenofibrate 360.84 Metabolism Hypocholesterolemic 04D07 Bumetanide 364.42 Metabolism Diuretic 04D08 Labetalol hydrochloride 364.88 Cardiovascular Antihypotensive 04D09 Cinnarizine 368.53 Allergology Antihistaminic 04D10 Methylprednisolone, 6-alpha 374.48 Endocrinology Anti-inflammatory 04D11 Quinidine hydrochloride 378.90 Cardiovascular Antiarrhythmic monohydrate 04E02 Fludrocortisone acetate 422.50 Dermatology Anti-inflammatory 04E03 Fenoterol hydrobromide 384.27 Neuromuscular Bronchodilator 04E04 Homochlorcyclizine 387.78 Allergology Antihistaminic dihydrochloride 04E05 Diethylcarbamazine citrate 391.42 Infectiology Antihelmintic

228 04E06 Chenodiol 392.58 Gastroenterology Cholagogue 04E07 Perhexiline maleate 393.57 Cardiovascular Antianginal 04E08 Oxybutynin chloride 393.96 Neuromuscular Antispastic 04E09 Spiperone 395.48 Central Nervous Antipsychotic System 04E10 Pyrilamine maleate 401.47 Allergology Antihistaminic 04E11 Sulfinpyrazone 404.49 Hematology Antiplatelet 04F02 Dantrolene sodium salt 336.24 Neuromuscular Muscle relaxant 04F03 Trazodone hydrochloride 408.33 Central Nervous Antidepressant System 04F04 Glafenine hydrochloride 409.27 Metabolism Analgesic 04F05 Pimethixene maleate 409.51 Allergology Antihistaminic 04F06 Pergolide mesylate 410.60 Central Nervous Antiparkinsonian System 04F07 Acemetacin 415.83 Metabolism Anti-inflammatory 04F08 Benzydamine hydrochloride 345.88 Central Nervous Analgesic System 04F09 Fipexide hydrochloride 425.32 Central Nervous Anti-fatigue System 04F10 Mifepristone 429.61 Endocrinology Abortifacient 04F11 Diperodon hydrochloride 433.94 Neuromuscular Local anaesthetic 04G02 Lisinopril 441.53 Cardiovascular Antihypertensive 04G03 Lincomycin hydrochloride 443.01 Infectiology Antibacterial 04G04 Telenzepine dihydrochloride 443.40 Gastroenterology Antiulcer 04G05 Econazole nitrate 444.70 Infectiology Antifungal 04G06 Bupivacaine hydrochloride 324.90 Neuromuscular Local anaesthetic 04G07 Clemastine fumarate 459.97 Allergology Antiemetic 04G08 Oxytetracycline dihydrate 496.48 Infectiology Antibacterial 04G09 Pimozide 461.56 Central Nervous Antipsychotic System 04G10 Amodiaquin dihydrochloride 464.82 Infectiology Anti-inflammatory dihydrate 04G11 Mebeverine hydrochloride 466.02 Neuromuscular Antispastic 04H02 Ifenprodil tartrate 475.54 Cardiovascular Vasodilator 04H03 Flunarizine dihydrochloride 477.43 Central Nervous Anticonvulsant System 04H04 Trifluoperazine 480.43 Central Nervous Antiemetic dihydrochloride System 04H05 Enalapril maleate 492.53 Cardiovascular Antihypertensive 04H06 Minocycline hydrochloride 493.95 Infectiology Antibacterial 04H07 Glibenclamide 494.01 Endocrinology Antidiabetic 04H08 Guanethidine sulfate 494.70 Central Nervous Antihypertensive System 04H09 Quinacrine dihydrochloride 508.92 Infectiology Antihelmintic hydrate 04H10 Clofilium tosylate 510.18 Cardiovascular Antiarrhythmic 04H11 Fluphenazine 510.45 Central Nervous Antipsychotic dihydrochloride System 05A02 Streptomycin sulfate 1457.40 Infectiology Antibacterial 05A03 Alfuzosin hydrochloride 425.92 Cardiovascular Vasodilator 05A04 Chlorpropamide 276.74 Endocrinology Antidiabetic 05A05 Phenylpropanolamine 187.67 Respiratory Antihypotensive hydrochloride 05A06 Ascorbic acid 176.13 Metabolism Anti-oxidant

229 05A07 Methyldopa (L,-) 211.22 Cardiovascular Antihypertensive 05A08 Cefoperazone dihydrate 681.71 Infectiology Antibacterial 05A09 Zoxazolamine 168.58 Metabolism Antigout 05A10 Tacrine hydrochloride 234.73 Central Nervous CNS Stimulant System 05A11 Bisoprolol fumarate 766.98 Cardiovascular Antianginal 05B02 Tremorine dihydrochloride 265.23 Central Nervous CNS Stimulant System 05B03 Practolol 266.34 Cardiovascular Antianginal 05B04 Zidovudine, AZT 267.25 Infectiology Antiviral 05B05 Sulfisoxazole 267.31 Infectiology Antibacterial 05B06 Zaprinast 271.28 Cardiovascular Erectile dysfunction treatment 05B07 Chlormezanone 273.74 Central Nervous Anxiolytic System 05B08 Procainamide hydrochloride 271.79 Cardiovascular Antiarrhythmic 05B09 N6-methyladenosine 281.27 Oncology Antineoplastic 05B10 Guanfacine hydrochloride 282.56 Cardiovascular Antihypertensive 05B11 Domperidone 425.92 Central Nervous Antiemetic System 05C02 Furosemide 330.75 Metabolism Antihypertensive 05C03 Methapyrilene hydrochloride 297.85 Allergology Antihistaminic 05C04 Desipramine hydrochloride 302.85 Central Nervous Antidepressant System 05C05 Clorgyline hydrochloride 308.64 Central Nervous Antidepressant System 05C06 Clenbuterol hydrochloride 313.66 Neuromuscular Antiasthmatic 05C07 Maprotiline hydrochloride 313.87 Central Nervous Antidepressant System 05C08 Thioguanosine 299.31 Metabolism Antineoplastic 05C09 Chlorprothixene 352.33 Central Nervous Antiemetic hydrochloride System 05C10 Ritodrine hydrochloride 323.82 Neuromuscular Tocolytic 05C11 Clozapine 326.83 Central Nervous Antiparkinsonian System 05D02 Chlorthalidone 338.77 Metabolism Antihypertensive 05D03 Dobutamine hydrochloride 337.85 Cardiovascular Analeptic 05D04 Moclobemide 268.75 Central Nervous Antidepressant System 05D05 Clopamide 345.85 Metabolism Antihypertensive 05D06 Hycanthone 356.49 Infectiology Antihelmintic 05D07 Adenosine 5'- 365.24 Cardiovascular Antiarrhythmic monophosphate monohydrate 05D08 Amoxicillin 365.41 Metabolism Antibacterial 05D09 Pemirolast potassium 266.31 Ophthalmology Anti-inflammatory 05D10 Dextromethorphan 370.33 Central Nervous Antitussive hydrobromide monohydrate System 05D11 Droperidol 379.44 Central Nervous Antipsychotic System 05E02 Bambuterol hydrochloride 403.91 Neuromuscular Bronchodilator 05E03 Betamethasone 392.47 Endocrinology Anti-inflammatory 05E04 Colchicine 399.45 Metabolism Antigout 05E05 Metergoline 403.53 Central Nervous Antiprolactin System

230 05E06 Brinzolamide 383.51 Metabolism Antiglaucoma 05E07 Ambroxol hydrochloride 414.57 Respiratory Expectorant 05E08 Benfluorex 351.37 Central Nervous Anorectic System 05E09 Bepridil hydrochloride 403.01 Cardiovascular Antianginal 05E10 Meloxicam 351.41 Metabolism Anti-inflammatory 05E11 Benzbromarone 424.09 Cardiovascular Antianginal 05F02 Ketotifen fumarate 425.51 Allergology Antihistaminic 05F03 Debrisoquin sulfate 448.55 Cardiovascular Antihypertensive 05F04 Amethopterin (R,S) 454.45 Immunology Anti-inflammatory 05F05 Methylergometrine maleate 455.52 Neuromuscular Hemostatic 05F06 Methiothepin maleate 472.63 Central Nervous Antipsychotic System 05F07 Clofazimine 473.41 Infectiology Antibacterial 05F08 Nafronyl oxalate 473.57 Cardiovascular Anti-ischemic 05F09 Bezafibrate 361.83 Metabolism Antilipemic 05F10 Nefazodone hydrochloride 506.48 Central Nervous Antidepressant System 05F11 Clebopride maleate 489.96 Central Nervous Antiemetic System 05G02 Lidoflazine 491.63 Cardiovascular Antianginal 05G03 Betaxolol hydrochloride 343.90 Cardiovascular Antiglaucoma 05G04 Nicardipine hydrochloride 516.00 Cardiovascular Antianginal 05G05 Probucol 516.86 Metabolism Antilipemic 05G06 Mitoxantrone 517.41 Oncology Antineoplastic dihydrochloride 05G07 GBR 12909 dihydrochloride 523.50 Central Nervous Antidepressant System 05G08 Carbetapentane citrate 525.60 Central Nervous Antispastic System 05G09 Dequalinium dichloride 527.59 Infectiology Antibacterial 05G10 Ketoconazole 531.44 Infectiology Antifungal 05G11 Fusidic acid sodium salt 538.71 Infectiology Antibacterial 05H02 Terbutaline hemisulfate 548.66 Respiratory Antiasthmatic 05H03 Ketanserin tartrate hydrate 563.54 Cardiovascular Antihypertensive 05H04 Hemicholinium bromide 574.36 Neuromuscular Curarizing 05H05 Kanamycin A sulfate 582.59 Infectiology Antibacterial 05H06 Amikacin hydrate 621.64 Infectiology Antibacterial 05H07 Etoposide 588.57 Oncology Antineoplastic 05H08 Clomiphene citrate (Z,E) 598.10 Endocrinology 05H09 Oxantel pamoate 820.95 Infectiology Antihelmintic 05H10 Prochlorperazine dimaleate 606.10 Central Nervous Antiemetic System 05H11 Hesperidin 610.57 Oncology Anti-haemorrhoids 06A02 Testosterone propionate 344.50 Endocrinology Anabolic 06A03 Haloprogin 361.40 Infectiology Antifungal 06A04 Thyroxine (L) 776.88 Endocrinology Antihypothyroid 06A05 Idebenone 338.45 Oncology Antineoplastic 06A06 Pepstatin A 685.91 Infectiology Antiviral 06A07 Delavirdine 456.57 Infectiology 06A08 Adamantamine fumarate 418.58 Infectiology Antiviral 06A09 Butoconazole nitrate 474.80 Infectiology Antibacterial 06A10 Amiodarone hydrochloride 681.78 Cardiovascular Antianginal

231 06A11 Amphotericin B 924.10 Infectiology Antibacterial 06B02 Androsterone 290.45 Endocrinology Anabolic 06B03 Amifostine 214.22 Diagnostic 06B04 Carbarsone 260.08 Infectiology Antiamebic 06B05 Amlodipine 408.89 Cardiovascular Antihypertensive 06B06 Modafinil 273.36 Central Nervous CNS Stimulant System 06B07 Bacampicillin hydrochloride 501.99 Infectiology Antibacterial 06B08 Lamivudine 229.26 Infectiology Antiviral 06B09 Biotin 244.31 Metabolism 06B10 Bisacodyl 361.40 Gastroenterology Laxative 06B11 Erlotinib 393.45 Oncology Antineoplastic 06C02 Suloctidil 337.57 Neuromuscular Antiplatelet 06C03 Zotepine 331.87 Central Nervous Antipsychotic System 06C04 Carisoprodol 260.34 Central Nervous Analgesic System 06C05 Cephalosporanic acid, 7- 272.28 Infectiology Antibacterial amino 06C06 Chicago sky blue 6B 992.82 Central Nervous System 06C07 Buflomedil hydrochloride 343.85 Cardiovascular Vasodilator 06C08 Dibenzepine hydrochloride 331.85 Central Nervous Antidepressant System 06C09 Roxatidine Acetate 384.91 Gastroenterology Antiulcer hydrochloride 06C10 Valacyclovir hydrochloride 360.80 Infectiology Antiviral 06C11 Cisapride 465.96 Gastroenterology Gastroprokinetic 06D02 Pefloxacine 333.37 Infectiology Antibacterial 06D03 Corticosterone 346.47 Endocrinology Anti-inflammatory 06D04 Cyanocobalamin 1355.40 Metabolism Analgesic 06D05 Cefadroxil 363.39 Infectiology Antibacterial 06D06 Cyclosporin A 1202.64 Immunology Immunosuppressant 06D07 Digitoxigenin 374.53 Cardiovascular Cardiotonic 06D08 Digoxin 780.96 Cardiovascular Cardiotonic 06D09 Doxorubicin hydrochloride 579.99 Infectiology Antibacterial 06D10 Carbimazole 186.23 Metabolism Antihyperthyroid 06D11 Epiandrosterone 290.45 Endocrinology Anabolic 06E02 Estradiol-17 beta 272.39 Endocrinology Antigonadotropin 06E03 Clobutinol hydrochloride 292.25 Central Nervous Antitussive System 06E04 Gabazine bromide 368.23 Central Nervous CNS Stimulant System 06E05 Oxcarbazepine 252.28 Central Nervous Anticonvulsant System 06E06 Cyclobenzaprine 311.86 Neuromuscular Muscle relaxant hydrochloride 06E07 Carteolol hydrochloride 328.84 Cardiovascular Antiglaucoma 06E08 Hydrocortisone base 362.47 Endocrinology Anti-inflammatory 06E09 Hydroxytacrine maleate 330.34 Central Nervous Anti-Alzheimer (R,S) System 06E10 Pilocarpine nitrate 271.28 Ophthalmology Antiglaucoma 06E11 Dicloxacillin sodium salt 510.33 Infectiology Antibacterial hydrate

232 06F02 Alizapride hydrochloride 351.84 Central Nervous Antiemetic System 06F03 Stanozolol 328.50 Endocrinology 06F04 Calcipotriene 412.62 Dermatology Antipsoriatic 06F05 Linezolid 337.35 Infectiology Antibacterial 06F06 Mebhydroline 1,5- 841.07 Allergology Antihistaminic naphtalenedisulfonate 06F07 Meclocycline sulfosalicylate 695.06 Infectiology Antibacterial 06F08 Meclozine dihydrochloride 463.88 Allergology Antiemetic 06F09 Melatonin 232.28 Central Nervous Anticonvulsant System 06F10 Butalbital 224.26 Central Nervous Hypnotic System 06F11 Dinoprost trometamol 475.63 Endocrinology Oxytocic 06G02 Tropisetron hydrochloride 320.82 Central Nervous Antiemetic System 06G03 Cefixime 453.46 Infectiology Antibacterial 06G04 Metrizamide 789.10 Diagnostic Contrastant 06G05 Quetiapine hemifumarate 883.11 Central Nervous Antipsychotic System 06G06 Tosufloxacin hydrochloride 440.81 Infectiology Antibacterial 06G07 Efavirenz 315.68 Infectiology Antiviral 06G08 Rifapentine 877.05 Infectiology Antibacterial 06G09 Neostigmine bromide 303.20 Diagnostic Anti-fatigue 06G10 Niridazole 214.20 Infectiology Antihelmintic 06G11 Ceforanide 519.56 Infectiology Antibacterial 06H02 Vatalanib 346.82 Oncology Antineoplastic 06H03 Itopride 358.44 Metabolism 06H04 Cefotetan 575.62 Infectiology Antibacterial 06H05 Fentiazac 329.81 Metabolism Anti-inflammatory 06H06 Brompheniramine maleate 435.32 Allergology Antihistaminic 06H07 Primaquine diphosphate 455.34 Infectiology Antimalarial 06H08 Progesterone 314.47 Endocrinology Progestogen 06H09 Felodipine 384.26 Neuromuscular Antianginal 06H10 Raclopride 347.24 Central Nervous System 06H11 Closantel 663.08 Infectiology Antihelmintic 07A02 Serotonin hydrochloride 212.68 Central Nervous CNS Stimulant System 07A03 Cefotiam hydrochloride 598.56 Infectiology Antibacterial 07A04 Rofecoxib 314.36 Metabolism Anti-inflammatory 07A05 Benperidol 381.45 Central Nervous Antipsychotic System 07A06 Cefaclor hydrate 385.83 Infectiology Antibacterial 07A07 Colistin sulfate 1253.54 Infectiology Antibacterial 07A08 Daunorubicin hydrochloride 563.99 Infectiology Antibacterial 07A09 Dosulepin hydrochloride 331.91 Central Nervous Antidepressant System 07A10 Ceftazidime pentahydrate 636.66 Infectiology Antibacterial 07A11 Iobenguane sulfate 373.17 Oncology Antineoplastic 07B02 Metixene hydrochloride 345.94 Central Nervous Antiparkinsonian System 07B03 Nitrofural 198.14 Infectiology Antibacterial 07B04 Omeprazole 345.42 Gastroenterology Antiulcer

233 07B05 Propylthiouracil 170.23 Metabolism Antihyperthyroid 07B06 Terconazole 532.47 Infectiology Antifungal 07B07 Tiaprofenic acid 260.31 Central Nervous Analgesic System 07B08 Vancomycin hydrochloride 1485.75 Infectiology Antibacterial 07B09 Artemisinin 282.34 Infectiology Antimalarial 07B10 Propafenone hydrochloride 377.92 Cardiovascular Antiarrhythmic 07B11 Ethamivan 223.27 Central Nervous Analeptic System 07C02 Vigabatrin hydrochloride 165.62 Central Nervous Anticonvulsant System 07C03 Biperiden hydrochloride 347.93 Central Nervous Antiparkinsonian System 07C04 Cetirizine dihydrochloride 461.82 Allergology Antihistaminic 07C05 Etifenin 322.36 Diagnostic Chemosensitizer 07C06 Metaproterenol sulfate, 520.60 Respiratory Bronchodilator orciprenaline sulfate 07C07 Sisomicin sulfate 545.61 Infectiology Antibacterial 07C08 Sibutramine hydrochloride 316.32 Central Nervous System 07C09 Acenocoumarol 353.33 Hematology Anticoagulant 07C10 Bromperidol 420.33 Central Nervous Antipsychotic System 07C11 Cyclizine hydrochloride 302.85 Allergology Antiemetic 07D02 Fluoxetine hydrochloride 345.80 Central Nervous Antidepressant System 07D03 Iohexol 821.15 Diagnostic Contrastant 07D04 Norcyclobenzaprine 261.37 Gastroenterology Antiulcer 07D05 Pyrazinamide 123.12 Infectiology Antibacterial 07D06 Trimethadione 143.14 Central Nervous Anticonvulsant System 07D07 Lovastatin 404.55 Metabolism Hypocholesterolemic 07D08 Nystatine 926.12 Infectiology Antifungal 07D09 Budesonide 430.55 Endocrinology Anti-inflammatory 07D10 Imipenem 299.35 Infectiology Antibacterial 07D11 Sulfasalazine 398.40 Infectiology Antibacterial 07E02 Lofexidine 259.14 Cardiovascular Antihypertensive 07E03 Thiostrepton 1664.92 Infectiology Antibacterial 07E04 Miglitol 207.23 Endocrinology Antidiabetic 07E05 Tiabendazole 201.25 Infectiology Antifungal 07E06 Rifampicin 822.96 Infectiology Antibacterial 07E07 Ethionamide 166.25 Infectiology Antibacterial 07E08 Tenoxicam 337.38 Central Nervous Analgesic System 07E09 Triflusal 248.16 Hematology Anticoagulant 07E10 Mesoridazine besylate 544.76 Central Nervous Antipsychotic System 07E11 Trolox 250.30 Metabolism Anti-oxidant 07F02 Pirenperone 393.47 Central Nervous System 07F03 Grepafloxacin 359.40 Infectiology 07F04 Phenacetin 179.22 Central Nervous Analgesic System 07F05 Atovaquone 366.85 Infectiology Antimalarial

234 07F06 Methoxamine hydrochloride 247.72 Cardiovascular Antihypotensive 07F07 (S)-(-)-Atenolol 266.34 Cardiovascular Antianginal 07F08 Piracetam 142.16 Central Nervous CNS Stimulant System 07F09 Phenindione 222.25 Hematology Anticoagulant 07F10 Thiocolchicoside 563.63 Central Nervous Antispastic System 07F11 Clorsulon 380.66 Infectiology Antihelmintic 07G02 Ciclopirox ethanolamine 268.36 Infectiology Antibacterial 07G03 Probenecid 285.36 Metabolism Antigout 07G04 Betahistine mesylate 328.41 Allergology Vasodilator 07G05 Tobramycin 467.52 Infectiology Antibacterial 07G06 Tetramisole hydrochloride 240.76 Immunology Antihelmintic 07G07 Pregnenolone 316.49 Endocrinology Anabolic 07G08 Molsidomine 242.24 Cardiovascular Antianginal 07G09 Chloroquine diphosphate 515.87 Metabolism Anti-inflammatory 07G10 Trimetazidine 339.26 Cardiovascular Antianginal dihydrochloride 07G11 Parthenolide 248.32 Metabolism Anti-inflammatory 07H02 Hexetidine 339.61 Infectiology Antifungal 07H03 Selegiline hydrochloride 223.75 Central Nervous Antiparkinsonian System 07H04 Pentamidine isethionate 592.69 Infectiology Antifungal 07H05 Tolazamide 311.41 Metabolism Antidiabetic 07H06 Nifuroxazide 275.22 Infectiology Antibacterial 07H07 Mirtazapine 265.36 Central Nervous Antidepressant System 07H08 Dirithromycin 835.09 Infectiology Antibacterial 07H09 Gliclazide 323.42 Metabolism Anticoagulant 07H10 DO 897/99 490.48 Central Nervous Antidepressant System 07H11 Prenylamine lactate 419.57 Cardiovascular Antianginal 08A02 Ziprasidone Hydrochloride 449.41 Central Nervous Antipsychotic System 08A03 Mevastatin 390.52 Cardiovascular Hypocholesterolemic 08A04 Pyridostigmine iodide 308.12 Central Nervous System 08A05 Pentobarbital 226.28 Central Nervous Anaesthetic System 08A06 Atropine sulfate 694.85 Ophthalmology Antispastic monohydrate 08A07 Eserine hemisulfate salt 648.78 Central Nervous Antiglaucoma System 08A08 Itraconazole 705.65 Infectiology Antifungal 08A09 Acarbose 645.62 Endocrinology Antidiabetic 08A10 Entacapone 305.29 Central Nervous Antiparkinsonian System 08A11 Nicotinamide 122.13 Dermatology 08B02 Tetracaïne hydrochloride 300.83 Neuromuscular 08B03 Mometasone furoate 521.44 Endocrinology Anti-inflammatory 08B04 Troglitazone 441.55 Metabolism Antidiabetic 08B05 Dacarbazine 182.19 Oncology Antineoplastic 08B06 Tenatoprazole 346.41 Metabolism Antiulcer

235 08B07 Acetopromazine maleate salt 442.54 Central Nervous Antiemetic System 08B08 Escitalopram oxalate 414.44 Central Nervous Antidepressant System 08B09 Ropinirole hydrochloride 296.84 Central Nervous Antiparkinsonian System 08B10 Lacidipine 455.56 Cardiovascular Antihypertensive 08B11 Argatroban 508.64 Hematology Anticoagulant 08C02 Reboxetine mesylate 409.51 Central Nervous Antidepressant System 08C03 Camylofine chlorhydrate 393.40 08C04 Papaverine hydrochloride 375.86 Cardiovascular Antispastic 08C05 Yohimbine hydrochloride 390.91 Cardiovascular Erectile dysfunction treatment 08C06 Voriconazole 349.32 Infectiology Antifungal 08C07 Alfacalcidol 400.65 Metabolism Antiosteoporetic 08C08 Cilostazol 369.47 Hematology Anticoagulant 08C09 Galanthamine hydrobromide 368.27 Central Nervous Analgesic System 08C10 Azelastine hydrochloride 418.37 Immunology Antihistaminic 08C11 Etretinate 354.49 Dermatology Antipsoriatic 08D02 Emedastine 534.57 Allergology Antihistaminic 08D03 Etofenamate 369.34 Metabolism Anti-inflammatory 08D04 Zaleplon 305.34 Central Nervous Hypnotic System 08D05 Diclofenac sodium 318.14 Central Nervous Anti-inflammatory System 08D06 Exemestane 296.41 Endocrinology Antineoplastic 08D07 Fomepizole 82.11 Metabolism 08D08 Temozolomide 194.15 Oncology Antineoplastic 08D09 Xylazine 220.34 Central Nervous Analgesic System 08D10 Celiprolol hydrochloride 415.96 Cardiovascular Antianginal 08D11 Zopiclone 388.82 Central Nervous Hypnotic System 08E02 Tranilast 327.34 Allergology Antiallergic 08E03 Tizanidine hydrochloride 290.18 Metabolism Muscle relaxant 08E04 Zafirlukast 575.69 Respiratory Antiasthmatic 08E05 Butenafine Hydrochloride 353.94 Infectiology Antifungal 08E06 Carbadox 262.23 Infectiology Antibacterial 08E07 Rimantadine Hydrochloride 215.77 Infectiology Antiviral 08E08 Eburnamonine (-) 294.40 Central Nervous Vasodilator System 08E09 Oxibendazol 249.27 Metabolism 08E10 Ipsapirone 401.49 Central Nervous System 08E11 Hydroxychloroquine sulfate 433.96 Metabolism Antimalarial 08F02 Loracarbef 349.78 Infectiology Antibacterial 08F03 Fenipentol 164.25 Metabolism Choleretic 08F04 Diosmin 608.56 Cardiovascular 08F05 Carbidopa 226.23 Central Nervous Antiparkinsonian System 08F06 (-)-Emtricitabine 247.25 Infectiology Antiviral 08F07 Demecarium bromide 716.60 Ophthalmology Antiglaucoma

236 08F08 Quipazine dimaleate salt 445.43 Central Nervous Antiemetic System 08F09 Acipimox 154.13 Metabolism Antilipemic 08F10 Diflorasone Diacetate 494.54 Endocrinology Anti-inflammatory 08F11 Acamprosate calcium 400.49 Central Nervous System 08G02 Mizolastine 432.50 Allergology 08G03 Amisulpride 369.49 Central Nervous Antipsychotic System 08G04 Pyridoxine hydrochloride 205.64 Metabolism 08G05 Mercaptopurine 152.18 Immunology Immunosuppressant 08G06 Cytarabine 243.22 Oncology Antineoplastic 08G07 Racecadotril 385.49 Gastroenterology Antidiarrheal 08G08 Folic acid 441.41 Metabolism 08G09 Benazepril hydrochloride 460.96 Cardiovascular Antihypertensive 08G10 Aniracetam 219.24 Central Nervous Anti-Alzheimer System 08G11 Dimethisoquin 308.85 Neuromuscular Antipruritic hydrochloride 08H02 Alendronate sodium 271.08 Metabolism Antiosteoporetic 08H03 Dipivefrin hydrochloride 387.91 Ophthalmology Antiglaucoma 08H04 Thiorphan 253.32 Gastroenterology Antidiarrheal 08H05 Tomoxetine hydrochloride 291.82 Central Nervous System 08H06 Aceclidine Hydrochloride 205.69 Ophthalmology Antiglaucoma 08H07 Penciclovir 253.26 Infectiology Antiviral 08H08 Levetiracetam 170.21 Central Nervous Anticonvulsant System 08H09 Dexfenfluramine 267.72 Central Nervous Anorectic hydrochloride System 08H10 Etoricoxib 358.85 Central Nervous Analgesic System 08H11 Sertindole 440.95 Central Nervous Antipsychotic System 09A02 Sulmazole 287.34 Cardiovascular Cardiotonic 09A03 Gefitinib 446.91 Oncology Antineoplastic 09A04 Flunisolide 434.51 Endocrinology Anti-inflammatory 09A05 N-Acetyl-DL-homocysteine 159.21 Respiratory Expectorant Thiolactone 09A06 Flurandrenolide 436.53 Dermatology Anti-inflammatory 09A07 Oxiconazole Nitrate 492.15 Infectiology Antifungal 09A08 Rebamipide 370.80 Metabolism Antiulcer 09A09 Nilvadipine 385.38 Cardiovascular Antianginal 09A10 Etanidazole 214.18 Oncology Antineoplastic 09A11 Pinaverium bromide 591.43 Neuromuscular Antispastic 09B02 Glimepiride 490.63 Endocrinology Antidiabetic 09B03 Picrotoxinin 292.29 Central Nervous Analeptic System 09B04 Mepenzolate bromide 420.35 Neuromuscular Antispastic 09B05 Benfotiamine 466.46 Metabolism 09B06 Halcinonide 454.97 Dermatology Anti-inflammatory 09B07 Lanatoside C 985.14 Cardiovascular Cardiotonic 09B08 Benzamil hydrochloride 356.22 Metabolism Antihypertensive

237 09B09 Suxibuzone 438.48 Central Nervous Analgesic System 09B10 6-Furfurylaminopurine 215.22 Dermatology 09B11 Avermectin B1a 873.10 Infectiology Antihelmintic 09C02 Pranlukast 481.52 Respiratory Antiasthmatic 09C03 D,L-Penicillamine 149.21 Central Nervous Analgesic System 09C04 Zileuton 236.29 Respiratory Antiasthmatic 09C05 Loratadine 382.89 Allergology Antihistaminic 09C06 Tetraethylenepentamine 371.61 Metabolism Antilipemic pentahydrochloride 09C07 Nisoldipine 388.42 Cardiovascular Antianginal 09C08 Acefylline 238.20 Central Nervous CNS Stimulant System 09C09 Acitretin 326.44 Dermatology Antipsoriatic 09C10 Zonisamide 212.23 Central Nervous Anticonvulsant System 09C11 Irsogladine maleate 372.17 Metabolism Antiulcer 09D02 Dydrogesterone 312.46 Endocrinology Progestogen 09D03 Sumatriptan succinate 413.50 Central Nervous Antimigraine System 09D04 Opipramol dihydrochloride 436.43 Central Nervous Antidepressant System 09D05 Nalidixic acid sodium salt 254.22 Infectiology Antibacterial 09D06 Oxacillin sodium 423.43 Infectiology Antibacterial 09D07 Beta-Escin 1131.28 Metabolism Antineoplastic 09D08 Thiamine hydrochloride 337.27 Immunology Immunostimulant 09D09 Tazobactam 300.29 Infectiology Antibacterial 09D10 Ibandronate sodium 341.22 Metabolism Antiosteoporetic 09D11 Warfarin 308.34 Hematology Anticoagulant 09E02 Pranoprofen 255.28 Metabolism Anti-inflammatory 09E03 Secnidazole 185.18 Infectiology Antiamebic 09E04 Pempidine tartrate 305.37 Cardiovascular Antihypotensive 09E05 Mirabegron 396.52 Neuromuscular 09E06 Ibutilide fumarate 885.25 Cardiovascular Antiarrhythmic 09E07 Tigecycline 585.66 Infectiology 09E08 Tramadol hydrochloride 299.84 Central Nervous Analgesic System 09E09 Estropipate 436.57 Endocrinology 09E10 Butylscopolammonium (n-) 440.38 Central Nervous Antispastic bromide System 09E11 Irinotecan hydrochloride 677.20 Oncology Antineoplastic trihydrate 09F02 Tylosin 916.12 Infectiology Antibacterial 09F03 Citalopram Hydrobromide 405.31 Central Nervous Antidepressant System 09F04 Promazine hydrochloride 320.89 Central Nervous Antipsychotic System 09F05 Sulfamerazine 264.31 Infectiology Antibacterial 09F06 Venlafaxine 277.41 Central Nervous Antidepressant System 09F07 Ethotoin 204.23 Central Nervous Anticonvulsant System 09F08 3-alpha-Hydroxy-5-beta- 290.45 Endocrinology androstan-17-one

238 09F09 Tetrahydrozoline 236.75 Cardiovascular Nasal Decongestant hydrochloride 09F10 Hexestrol 270.37 Endocrinology Antineoplastic 09F11 Cefmetazole sodium salt 493.52 Infectiology Antibacterial 09G02 Trihexyphenidyl-D, L 337.94 Central Nervous Antiparkinsonian Hydrochloride System 09G03 Succinylsulfathiazole 355.39 Infectiology Antibacterial 09G04 Famprofazone 377.53 Central Nervous Analgesic System 09G05 Bromopride 344.25 Central Nervous Antiemetic System 09G06 Methyl benzethonium 462.12 Infectiology Antibacterial chloride 09G07 Chlorcyclizine 337.30 Allergology Antiemetic hydrochloride 09G08 Diphenylpyraline 317.86 Allergology Antihistaminic hydrochloride 09G09 Benzethonium chloride 448.09 Infectiology Antibacterial 09G10 Trioxsalen 228.25 Dermatology 09G11 Doxofylline 266.26 Respiratory Bronchodilator 09H02 Sulfabenzamide 276.32 Infectiology Antibacterial 09H03 Benzocaine 165.19 Neuromuscular Local Anaesthetic 09H04 Dipyrone 333.34 Central Nervous Analgesic System 09H05 Isosorbide dinitrate 236.14 Cardiovascular Antianginal 09H06 Sulfachloropyridazine 284.73 Infectiology Antibacterial 09H07 Pramoxine hydrochloride 329.87 Neuromuscular Local Anaesthetic 09H08 Finasteride 372.56 Endocrinology Anti-alopecia 09H09 Fluorometholone 376.47 Endocrinology Anti-inflammatory 09H10 Cephalothin sodium salt 418.43 Infectiology Antibacterial 09H11 Cefuroxime sodium salt 446.37 Infectiology Antibacterial 10A02 Althiazide 383.90 Metabolism Antihypertensive 10A03 Isopyrin hydrochloride 281.79 Central Nervous Analgesic System 10A04 Phenethicillin potassium salt 402.52 Infectiology Antibacterial 10A05 Sulfamethoxypyridazine 280.31 Infectiology Antibacterial 10A06 Deferoxamine mesylate 656.80 Diagnostic Chelating 10A07 Mephentermine hemisulfate 424.61 Cardiovascular Antihypotensive 10A08 Liranaftate 328.44 Infectiology Antifungal 10A09 Sulfadimethoxine 310.33 Infectiology Antibacterial 10A10 Sulfanilamide 172.21 Infectiology Antibacterial 10A11 Balsalazide Sodium 401.29 Gastroenterology Anti-inflammatory 10B02 Sulfaquinoxaline sodium salt 322.32 Infectiology Antibacterial 10B03 Streptozotocin 265.22 Oncology Antineoplastic 10B04 Metoprolol-(+,-) (+)-tartrate 684.83 Cardiovascular Antiarrhythmic salt 10B05 Flumethasone 410.46 Metabolism Anti-inflammatory 10B06 Flecainide acetate 474.40 Cardiovascular Antiarrhythmic 10B07 Cefazolin sodium salt 476.49 Metabolism Antibacterial 10B08 Trimetozine 281.31 Central Nervous Sedative System 10B09 Folinic acid calcium salt 511.51 Hematology Antianemic 10B10 Levonordefrin 183.21 Cardiovascular Vasoconstrictor 10B11 Ebselen 274.18 Metabolism Anti-inflammatory

239 10C02 Nadide 663.44 Metabolism 10C03 Sulfamethizole 270.33 Metabolism Antibacterial 10C04 Medrysone 344.50 Metabolism Anti-inflammatory 10C05 Flunixin meglumine 491.47 Central Nervous Analgesic System 10C06 Spiramycin 842.07 Metabolism Antibacterial 10C07 Glycopyrrolate 398.34 Gastroenterology Antispastic 10C08 Aprepitant 534.44 Metabolism Antiemetic 10C09 Monensin sodium salt 692.87 Infectiology Antibacterial 10C10 Isoetharine mesylate salt 335.42 Respiratory Bronchodilator 10C11 Mevalonic-D, L acid lactone 130.14 Cardiovascular Antilipemic 10D02 Terazosin hydrochloride 423.90 Cardiovascular Antihypertensive 10D03 Phenazopyridine 249.70 Central Nervous Analgesic hydrochloride System 10D04 Demeclocycline 501.32 Metabolism Antibacterial hydrochloride 10D05 Fenoprofen calcium salt 558.65 Metabolism Anti-inflammatory dihydrate 10D06 Piperacillin sodium salt 539.55 Metabolism Antibacterial 10D07 Diethylstilbestrol 268.36 Endocrinology 10D08 Chlorotrianisene 380.88 Endocrinology Antineoplastic 10D09 Ribostamycin sulfate salt 552.56 Metabolism Antibacterial 10D10 Methacholine chloride 195.69 Diagnostic 10D11 Pipenzolate bromide 434.38 Gastroenterology Antispastic 10E02 Butamben 193.25 Central Nervous Anaesthetic System 10E03 Sulfapyridine 249.29 Metabolism Antibacterial 10E04 Meclofenoxate 294.18 Central Nervous CNS Stimulant hydrochloride System 10E05 Furaltadone hydrochloride 360.76 Infectiology Antibacterial 10E06 Ethoxyquin 217.31 Metabolism Antifungal 10E07 Tinidazole 247.27 Infectiology Antiamebic 10E08 Guanadrel sulfate 524.64 Cardiovascular Antihypertensive 10E09 Vidarabine 267.25 Metabolism Antiviral 10E10 Sulfameter 280.31 Metabolism Antibacterial 10E11 Isopropamide iodide 480.44 Metabolism Antiulcer 10F02 Alclometasone dipropionate 521.06 Metabolism Anti-inflammatory 10F03 Leflunomide 270.21 Immunology Immunosuppressant 10F04 Norgestrel-(-)-D 312.46 Endocrinology Contraceptive 10F05 Fluocinonide 494.54 Metabolism Anti-inflammatory 10F06 Sulfamethazine sodium salt 300.32 Metabolism Antibacterial 10F07 Guaifenesin 198.22 Respiratory Bronchodilator 10F08 Alexidine dihydrochloride 581.73 Infectiology Antibacterial 10F09 Proadifen hydrochloride 389.97 Neuromuscular Local Anaesthetic 10F10 Zomepirac sodium salt 313.72 Metabolism Anti-inflammatory 10F11 Cinoxacin 262.22 Metabolism Antibacterial 10G02 Clobetasol propionate 466.98 Metabolism Anti-inflammatory 10G03 Podophyllotoxin 414.42 Metabolism Antiviral 10G04 Clofibric acid 214.65 Metabolism Antilipemic 10G05 Bendroflumethiazide 421.42 Cardiovascular Antihypertensive 10G06 Dicumarol 336.30 Hematology Anticoagulant 10G07 Methimazole 114.17 Endocrinology

240 10G08 Merbromin 750.66 Infectiology Antibacterial 10G09 Hexylcaine hydrochloride 297.83 Dermatology Anaesthetic 10G10 Drofenine hydrochloride 353.94 Neuromuscular Antispastic 10G11 Cycloheximide 281.35 Infectiology Antibacterial 10H02 (R) -Naproxen sodium salt 252.25 Metabolism Anti-inflammatory 10H03 Propidium iodide 668.41 Infectiology Antibacterial 10H04 Cloperastine hydrochloride 366.33 Respiratory Antitussive 10H05 Eucatropine hydrochloride 327.85 Neuromuscular Antiglaucoma 10H06 Isocarboxazid 231.26 Central Nervous Antidepressant System 10H07 Lithocholic acid 376.58 Metabolism Cholagogue 10H08 Methotrimeprazine maleat 444.55 Central Nervous Analgesic salt System 10H09 Dienestrol 266.34 Endocrinology 10H10 Pridinol methanesulfonate 391.53 Central Nervous Antiparkinsonian salt System 10H11 Amrinone 187.20 Cardiovascular 11A02 Carbinoxamine maleate salt 406.87 Allergology Antihistaminic 11A03 Methazolamide 236.27 Metabolism Antiglaucoma 11A04 Pyrithyldione 167.21 Central Nervous Hypnotic System 11A05 Spectinomycin 405.28 Metabolism Antibacterial dihydrochloride 11A06 Piromidic acid 288.31 Metabolism Antibacterial 11A07 Trimipramine maleate salt 410.52 Central Nervous Antidepressant System 11A08 Chloropyramine 326.27 Allergology Antihistaminic hydrochloride 11A09 Furazolidone 225.16 Metabolism 11A10 Dichlorphenamide 305.16 Ophthalmology Antiglaucoma 11A11 Sulconazole nitrate 460.77 Metabolism Antifungal 11B02 Auranofin 678.49 Metabolism Analgesic 11B03 Cromolyn disodium salt 512.34 Allergology Antiasthmatic 11B04 Bucladesine sodium salt 491.38 Cardiovascular 11B05 Cefsulodin sodium salt 554.54 Metabolism Antibacterial 11B06 Fosfosal 218.10 Central Nervous Analgesic System 11B07 Suprofen 260.31 Central Nervous Analgesic System 11B08 Deflazacort 441.53 Immunology Anti-inflammatory 11B09 Nadolol 309.41 Cardiovascular Antianginal 11B10 Moxalactam disodium salt 564.44 Metabolism Antibacterial 11B11 Aminophylline 420.43 Cardiovascular Bronchodilator 11C02 Azlocillin sodium salt 483.48 Metabolism Antibacterial 11C03 Clidinium bromide 432.36 Neuromuscular Antispastic 11C04 Sulfamonomethoxine 280.31 Metabolism Antibacterial 11C05 Benzthiazide 431.94 Cardiovascular Antihypertensive 11C06 Trichlormethiazide 380.66 Cardiovascular Antihypertensive 11C07 Oxalamine citrate salt 437.45 Central Nervous Anti-inflammatory System 11C08 Propantheline bromide 448.40 Neuromuscular Antispastic 11C09 Viloxazine hydrochloride 273.76 Central Nervous Antidepressant System

241 11C10 Dimethadione 129.12 Central Nervous Anticonvulsant System 11C11 Ethaverine hydrochloride 431.96 Central Nervous Antispastic System 11D02 Butacaine 306.45 Dermatology Anaesthetic 11D03 Cefoxitin sodium salt 449.44 Metabolism Antibacterial 11D04 Ifosfamide 261.09 Oncology Antineoplastic 11D05 Novobiocin sodium salt 634.62 Metabolism Antibacterial 11D06 Zolmitriptan 287.36 Central Nervous System 11D07 Indoprofen 281.31 Central Nervous Analgesic System 11D08 Carbenoxolone disodium salt 614.74 Metabolism Antiulcer 11D09 Iocetamic acid 613.96 Diagnostic Contrastant 11D10 Ganciclovir 255.24 Metabolism Antiviral 11D11 Ethopropazine hydrochloride 348.94 Central Nervous Antiparkinsonian System 11E02 Olanzapine 312.44 Central Nervous Antipsychotic System 11E03 Trimeprazine tartrate 747.00 Allergology Antihistaminic 11E04 Nafcillin sodium salt 454.48 Metabolism Antibacterial monohydrate 11E05 Procyclidine hydrochloride 323.91 Central Nervous Antiparkinsonian System 11E06 Amiprilose hydrochloride 341.84 Immunology Immunomodulator 11E07 Ethynylestradiol 3-methyl 310.44 Endocrinology ether 11E08 (-) -Levobunolol 327.85 Ophthalmology Antiglaucoma hydrochloride 11E09 Iodixanol 1550.20 Diagnostic Contrastant 11E10 Clinafloxacin 365.79 Infectiology Antibacterial 11E11 Equilin 268.36 Endocrinology 11F02 Paroxetine Hydrochloride 365.84 Central Nervous Antidepressant System 11F03 Nylidrin 299.42 Cardiovascular Vasodilator 11F04 Liothyronine 650.98 Endocrinology 11F05 Roxithromycin 837.07 Metabolism Antibacterial 11F06 Beclomethasone 521.06 Metabolism Anti-inflammatory dipropionate 11F07 Tolmetin sodium salt 315.30 Metabolism Anti-inflammatory dihydrate 11F08 (+) -Levobunolol 327.85 Ophthalmology Antiglaucoma hydrochloride 11F09 Doxazosin mesylate 547.59 Cardiovascular Antihypertensive 11F10 Fluvastatin sodium salt 433.46 Cardiovascular Antilipemic 11F11 Methylhydantoin-5-(L) 114.10 Central Nervous Anticonvulsant System 11G02 Gabapentin 171.24 Central Nervous Anticonvulsant System 11G03 Raloxifene hydrochloride 510.06 Endocrinology 11G04 Ciclesonide 540.70 Respiratory 11G05 Methylhydantoin-5-(D) 114.10 11G06 Simvastatin 418.58 Cardiovascular Antilipemic 11G07 Azacytidine-5 244.21 Oncology Antineoplastic 11G08 Paromomycin sulfate 713.72 Metabolism Antiamebic

242 11G09 Acetaminophen 151.17 Central Nervous Analgesic System 11G10 Phthalylsulfathiazole 403.44 Metabolism Antibacterial 11G11 Luteolin 286.24 Respiratory Expectorant 11H02 Iopamidol 777.09 Diagnostic Contrastant 11H03 Iopromide 791.12 Diagnostic Contrastant 11H04 Theophylline monohydrate 198.18 Cardiovascular Bronchodilator 11H05 Theobromine 180.17 Cardiovascular Bronchodilator 11H06 Reserpine 608.69 Central Nervous Antipsychotic System 11H07 Bicalutamide 430.38 Endocrinology Antineoplastic 11H08 Scopolamine hydrochloride 339.82 Central Nervous Antiemetic System 11H09 Ioversol 807.12 Diagnostic Contrastant 11H10 Rabeprazole Sodium salt 382.44 Metabolism Antiulcer 11H11 Carbachol 182.65 Cardiovascular Antihypertensive 12A02 Niacin 123.11 Cardiovascular Antilipemic 12A03 Bemegride 155.20 Central Nervous CNS stimulant System 12A04 Digoxigenin 390.52 Diagnostic 12A05 Meglumine 195.22 Metabolism Antileishmanial 12A06 Dolasetron mesilate 438.50 Central Nervous Antiemetic System 12A07 Clioquinol 305.50 Metabolism Antiamebic 12A08 Oxybenzone 228.25 Dermatology 12A09 Promethazine hydrochloride 320.89 Allergology Antihistaminic 12A10 Diacerein 368.30 Immunology Antiarthritic 12A11 Esmolol hydrochloride 331.84 Cardiovascular Antiarrhythmic 12B02 Cortisol acetate 404.51 Metabolism Anti-inflammatory 12B03 Flubendazol 313.29 Metabolism 12B04 Felbinac 212.25 Central Nervous Analgesic System 12B05 Butylparaben 194.23 Metabolism Antifungal 12B06 Aminohippuric acid 194.19 Diagnostic 12B07 N-Acetyl-L-leucine 173.21 Central Nervous Antivertigo System 12B08 Pipemidic acid 303.32 Metabolism Antibacterial 12B09 Dioxybenzone 244.25 Dermatology 12B10 Adrenosterone 300.40 Endocrinology 12B11 Methylatropine nitrate 366.42 Neuromuscular Antispastic 12C02 Hymecromone 176.17 Metabolism Muscle relaxant 12C03 Abacavir Sulfate 670.76 Infectiology Antiviral 12C04 Diloxanide furoate 328.15 Metabolism Antiamebic 12C05 Metyrapone 226.28 Endocrinology 12C06 Urapidil hydrochloride 423.95 Cardiovascular Antihypertensive 12C07 Fluspirilen 475.59 Central Nervous Antipsychotic System 12C08 S-(+)-ibuprofen 206.29 Central Nervous Analgesic System 12C09 Ethynodiol diacetate 384.52 Endocrinology Contraceptive 12C10 Nabumetone 228.29 Central Nervous Analgesic System 12C11 Nisoxetine hydrochloride 307.82 Central Nervous Antidepressant System

243 12D02 (+)-Isoproterenol (+)- 361.35 Respiratory Antiasthmatic bitartrate salt 12D03 Monobenzone 200.24 Dermatology 12D04 2- 172.21 Metabolism Diuretic Aminobenzenesulfonamide 12D05 Estrone 270.37 Endocrinology 12D06 Lorglumide sodium salt 481.40 Metabolism Antiulcer 12D07 Nitrendipine 360.37 Cardiovascular Antihypertensive 12D08 Flurbiprofen 244.27 Central Nervous Analgesic System 12D09 Nimodipine 418.45 Cardiovascular Vasodilator 12D10 Bacitracin 1422.73 Metabolism Antibacterial 12D11 L(-)-vesamicol 295.86 Neuromuscular hydrochloride 12E02 Nizatidine 331.46 Metabolism Antiulcer 12E03 Thioperamide maleate 408.52 Central Nervous Antiemetic System 12E04 Xamoterol hemifumarate 794.86 Cardiovascular 12E05 Rolipram 275.35 Central Nervous Antidepressant System 12E06 Thonzonium bromide 591.73 Dermatology Antiseptic 12E07 Idazoxan hydrochloride 240.69 Central Nervous Antiparkinsonian System 12E08 Quinapril hydrochloride 474.99 Cardiovascular Antihypertensive 12E09 Nilutamide 317.23 Oncology Antineoplastic 12E10 Ketorolac tromethamine 376.41 Central Nervous Analgesic System 12E11 Protriptyline hydrochloride 299.85 Central Nervous Antidepressant System 12F02 Propofol 178.28 Central Nervous Anaesthetic System 12F03 S(-)Eticlopride 377.31 Central Nervous hydrochloride System 12F04 Primidone 218.26 Central Nervous Anticonvulsant System 12F05 Flucytosine 129.09 Metabolism Antifungal 12F06 (-)-MK 801 hydrogen 337.38 Central Nervous Anticonvulsant maleate System 12F07 Bephenium 443.55 Metabolism hydroxynaphthoate 12F08 Dehydroisoandosterone 3- 330.47 Endocrinology acetate 12F09 Benserazide hydrochloride 293.71 Central Nervous Antiparkinsonian System 12F10 Iodipamide 1139.77 Diagnostic Contrastant 12F11 Allopurinol 136.11 Metabolism 12G02 Pentetic acid 393.35 Oncology Chelating 12G03 Bretylium tosylate 414.36 Cardiovascular Anaesthetic 12G04 Pralidoxime chloride 172.62 Neuromuscular 12G05 Phenoxybenzamine 340.30 Cardiovascular Antihypertensive hydrochloride 12G06 Salmeterol 415.58 Respiratory Bronchodilator 12G07 Altretamine 210.28 Oncology Antineoplastic 12G08 Prazosin hydrochloride 419.87 Cardiovascular Antihypertensive 12G09 Timolol maleate salt 432.50 Cardiovascular Antianginal

244 12G10 (+,-)-Octopamine 189.64 Cardiovascular hydrochloride 12G11 Stavudine 224.22 Infectiology Antiviral 12H02 Crotamiton 203.29 Dermatology Antipruritic 12H03 Toremifene 405.97 Endocrinology Antineoplastic 12H04 (R)-(+)-Atenolol 266.34 Cardiovascular Antianginal 12H05 Tyloxapol 997.42 Respiratory Mucolytic 12H06 Florfenicol 358.22 Metabolism Antibacterial 12H07 Megestrol acetate 384.52 Endocrinology Antineoplastic 12H08 Deoxycorticosterone 330.47 Endocrinology Anti-inflammatory 12H09 Urosiol 392.58 Metabolism 12H10 Proparacaine hydrochloride 330.86 Central Nervous Anaesthetic System 12H11 Aminocaproic acid 131.18 Allergology Antifibrinolytic 13A02 Denatonium benzoate 446.59 Neuromuscular 13A03 Canrenone 340.47 Endocrinology Diuretic 13A04 Enilconazole 297.19 Metabolism Antifungal 13A05 Methacycline hydrochloride 478.89 Metabolism Antibacterial 13A06 Floxuridine 246.20 Oncology Antineoplastic 13A07 Sotalol hydrochloride 308.83 Cardiovascular Antianginal 13A08 Gestrinone 308.42 Endocrinology Contraceptive 13A09 Decamethonium bromide 418.30 Neuromuscular Muscle relaxant 13A10 Darifenacin hydrobromide 507.48 Neuromuscular 13A11 Indatraline hydrochloride 328.67 Central Nervous Antidepressant System 13B02 Remoxipride Hydrochloride 407.74 Central Nervous Antipsychotic System 13B03 THIP Hydrochloride 176.60 Central Nervous Sedative System 13B04 Pirlindole mesylate 322.43 Central Nervous Antidepressant System 13B05 Pronethalol hydrochloride 265.79 Cardiovascular Antianginal 13B06 Naftopidil dihydrochloride 465.42 Cardiovascular Antihypertensive 13B07 Tracazolate hydrochloride 340.86 Central Nervous Anticonvulsant System 13B08 Zardaverine 268.22 Respiratory Bronchodilator 13B09 Memantine Hydrochloride 215.77 Central Nervous Anti-Alzheimer System 13B10 Ozagrel hydrochloride 264.71 Cardiovascular Antianginal 13B11 Piribedil hydrochloride 334.81 Cardiovascular Antiparkinsonian 13C02 Nitrocaramiphen 370.88 Central Nervous hydrochloride System 13C03 Nandrolone 274.41 Endocrinology Antianemic 13C04 Dimaprit dihydrochloride 234.19 Metabolism 13C05 Oxfendazol 315.35 Metabolism 13C06 Guaiacol 124.14 Respiratory Expectorant 13C07 Proscillaridin A 530.66 Cardiovascular 13C08 Pramipexole dihydrochloride 284.25 Central Nervous Antiparkinsonian System 13C09 Norgestimate 369.51 Endocrinology 13C10 Chlormadinone acetate 404.94 Endocrinology Antineoplastic 13C11 Phenylbutazone 308.38 Metabolism Anti-inflammatory 13D02 Gliquidone 527.64 Endocrinology Antidiabetic 13D03 Pizotifen malate 429.54 Allergology Antihistaminic

245 13D04 Ribavirin 244.21 Metabolism Antiviral 13D05 Cyclopenthiazide 379.89 Cardiovascular Antihypertensive 13D06 Fluvoxamine maleate 434.42 Central Nervous Antidepressant System 13D07 Prothionamide 180.27 Infectiology Antibacterial 13D08 Fluticasone propionate 500.58 Cardiovascular Anti-inflammatory 13D09 Zuclopenthixol 473.90 Central Nervous Antipsychotic dihydrochloride System 13D10 Proguanil hydrochloride 290.20 Metabolism Antimalarial 13D11 Lymecycline 602.65 Metabolism Antibacterial 13E02 Alfadolone acetate 390.52 Central Nervous Anaesthetic System 13E03 Alfaxalone 332.49 Central Nervous Anaesthetic System 13E04 Azapropazone 300.36 Central Nervous Analgesic System 13E05 Meptazinol hydrochloride 269.82 Central Nervous Analgesic System 13E06 Apramycin 539.59 Metabolism Antibacterial 13E07 Darunavir 547.68 Infectiology 13E08 Fursultiamine Hydrochloride 435.01 Metabolism Anti-Alzheimer 13E09 Gabexate mesilate 417.48 Haematology Anticoagulant 13E10 Pivampicillin 463.56 Metabolism Antibacterial 13E11 Lodoxamide 311.64 Allergology Antihistaminic 13F02 Flucloxacillin sodium 475.86 Metabolism Antibacterial 13F03 Trapidil 205.26 Cardiovascular Vasodilator 13F04 Deptropine citrate 525.60 Allergology Antihistaminic 13F05 Sertraline 306.24 Central Nervous Antidepressant System 13F06 Ethamsylate 263.31 Cardiovascular Antiplatelet 13F07 Moxonidine 241.68 Cardiovascular Antihypertensive 13F08 Etilefrine hydrochloride 217.70 Cardiovascular Vasoconstrictor 13F09 Alprostadil 354.49 Cardiovascular Erectile dysfunction treatment 13F10 Tribenoside 478.59 Cardiovascular 13F11 Rimexolone 370.54 Metabolism Anti-inflammatory 13G02 Isradipine 371.40 Cardiovascular Antianginal 13G03 Nifekalant 405.46 Cardiovascular Antiarrhythmic 13G04 Isometheptene mucate 492.66 Cardiovascular Antimigraine 13G05 Nifurtimox 287.30 Metabolism 13G06 Letrozole 285.31 Oncology Antineoplastic 13G07 Arbutin 272.26 Metabolism Antibacterial 13G08 Tocainide hydrochloride 228.72 Cardiovascular Anaesthetic 13G09 Benzathine benzylpenicillin 941.14 Metabolism Antibacterial 13G10 Risperidone 410.50 Central Nervous Antipsychotic System 13G11 Torsemide 348.43 Cardiovascular Antihypertensive 13H02 Halofantrine hydrochloride 536.90 Metabolism Antimalarial 13H03 Articaine hydrochloride 320.84 Central Nervous Anaesthetic System 13H04 Nomegestrol acetate 370.49 Endocrinology Contraceptive 13H05 Pancuronium bromide 732.69 Neuromuscular Muscle relaxant 13H06 Molindone hydrochloride 312.84 Central Nervous Antipsychotic System

246 13H07 Alcuronium chloride 737.82 Neuromuscular Muscle relaxant 13H08 Zalcitabine 211.22 Metabolism Antiviral 13H09 Methyldopate hydrochloride 275.73 Cardiovascular Antihypertensive 13H10 Levocabastine hydrochloride 456.99 Allergology Antihistaminic 13H11 Pyrvinium pamoate 1151.43 Metabolism 14A02 Etomidate 244.30 Central Nervous Anaesthetic System 14A03 Tridihexethyl chloride 353.98 Neuromuscular Antispastic 14A04 Penbutolol sulfate 680.95 Cardiovascular Antianginal 14A05 Prednicarbate 488.58 Metabolism Anti-Inflammatory 14A06 Sertaconazole nitrate 500.79 Metabolism Antibacterial 14A07 Repaglinide 452.60 Endocrinology Antidiabetic 14A08 Piretanide 362.41 Cardiovascular Antihypertensive 14A09 Piperacetazine 410.58 Central Nervous Antipsychotic System 14A10 Oxyphenbutazone 324.38 Metabolism Anti-inflammatory 14A11 Quinethazone 289.74 Cardiovascular Antihypertensive 14B02 Moricizine hydrochloride 463.99 Cardiovascular Antiarrhythmic 14B03 Iopanoic acid 570.94 Diagnostic Contrastant 14B04 Pivmecillinam hydrochloride 476.04 Metabolism Antibacterial 14B05 Levopropoxyphene 547.72 Central Nervous Analgesic napsylate System 14B06 Piperidolate hydrochloride 359.90 Neuromuscular Antispastic 14B07 Trifluridine 296.20 Metabolism Antiviral 14B08 Oxprenolol hydrochloride 301.82 Cardiovascular Antianginal 14B09 Ondansetron Hydrochloride 329.83 Central Nervous Antianemic System 14B10 Propoxycaine hydrochloride 330.86 Central Nervous Anaesthetic System 14B11 Oxaprozin 293.33 Central Nervous Analgesic System 14C02 Phensuximide 189.22 Central Nervous Anticonvulsant System 14C03 Ioxaglic acid 1268.89 Diagnostic Contrastant 14C04 Naftifine hydrochloride 323.87 Infectiology Antifungal 14C05 Meprylcaine hydrochloride 271.79 Neuromuscular Local anaesthetic 14C06 Milrinone 211.23 Cardiovascular Vasodilator 14C07 Methantheline bromide 420.35 Neuromuscular Antispastic 14C08 Ticarcillin sodium 406.41 Infectiology Antibacterial 14C09 Thiethylperazine dimalate 667.80 Central Nervous Antiemetic System 14C10 Mesalamine 153.14 Metabolism Anti-inflammatory 14C11 Vorinostat 264.33 Oncology Antineoplastic 14D02 Imidurea 388.30 Infectiology Antifungal 14D03 Lansoprazole 369.37 Metabolism Antiulcer 14D04 Bethanechol chloride 196.68 Metabolism 14D05 Cyproterone acetate 416.95 Endocrinology Antineoplastic 14D06 (R)-Propranolol 295.81 Cardiovascular Antianginal hydrochloride 14D07 Ciprofibrate 289.16 Metabolism Hypocholesterolemic 14D08 Formestane 302.42 Endocrinology Antineoplastic 14D09 Benzylpenicillin sodium 356.38 Infectiology Antibacterial 14D10 Methicillin sodium 402.40 Oncology

247 14D11 Methiazole 265.34 Infectiology Antihelmintic 14E02 (S)-propranolol 295.81 Cardiovascular Antianginal hydrochloride 14E03 (-)-Eseroline fumarate salt 334.38 Central Nervous Analgesic System 14E04 Isosorbide mononitrate 191.14 Cardiovascular Antianginal 14E05 Levalbuterol hydrochloride 275.78 Respiratory Antiasthmatic 14E06 Topiramate 339.37 Central Nervous Anticonvulsant System 14E07 D-cycloserine 102.09 Infectiology Antibacterial 14E08 Nelarabine 297.27 Infectiology 14E09 (+,-)-Synephrine 167.21 Cardiovascular Vasoconstrictor 14E10 (S)-(-)-Cycloserine 102.09 Infectiology Antibacterial 14E11 Homosalate 262.35 Dermatology Radioprotectant 14F02 Spaglumic acid 304.26 Allergology Antiallergic 14F03 Ranolazine 427.55 Cardiovascular Antianginal 14F04 Misoprostol 382.55 Metabolism Antiulcer 14F05 Sulfadoxine 310.33 Infectiology Antibacterial 14F06 Cyclopentolate 327.85 Metabolism hydrochloride 14F07 Estriol 288.39 Endocrinology 14F08 (-)-Isoproterenol 247.72 Cardiovascular Bronchodilator hydrochloride 14F09 Sarafloxacin 385.37 Infectiology Antibacterial 14F10 Nialamide 298.35 Central Nervous Antidepressant System 14F11 Toltrazuril 425.39 Infectiology Anticoccidial 14G02 Perindopril 368.48 Cardiovascular Antihypertensive 14G03 Fexofenadine hydrochloride 538.13 Allergology Antihistaminic 14G04 4-aminosalicylic acid 153.14 Infectiology Antibacterial 14G05 Clonixin Lysinate 408.89 Central Nervous Analgesic System 14G06 Verteporfin 718.81 Ophthalmology 14G07 Meropenem 383.47 Infectiology Antibacterial 14G08 Ramipril 416.52 Cardiovascular Antihypertensive 14G09 Mephenytoin 218.26 Central Nervous Anticonvulsant System 14G10 Rifabutin 847.03 Infectiology Antibacterial 14G11 Parbendazole 247.30 Infectiology 14H02 Mecamylamine 203.76 Cardiovascular Antihypertensive hydrochloride 14H03 Procarbazine hydrochloride 257.77 Oncology Antineoplastic 14H04 Viomycin sulfate 783.78 Infectiology Antibacterial 14H05 Saquinavir mesylate 766.96 Immunology Antiviral 14H06 Ronidazole 200.16 Infectiology Antibacterial 14H07 Dorzolamide hydrochloride 360.90 Cardiovascular Antiglaucoma 14H08 Azaperone 327.41 Central Nervous Antipsychotic System 14H09 Cefepime hydrochloride 517.03 Infectiology Antibacterial 14H10 Clocortolone pivalate 495.04 Endocrinology Anti-inflammatory 14H11 Nadifloxacin 360.39 Infectiology Antibacterial 15A02 Buspirone hydrochloride 421.97 Central Nervous System 15A03 Anastrozole 293.37 Oncology Antineoplastic

248 15A04 Doxycycline hydrochloride 480.91 Metabolism Antibacterial 15A05 Sulbactam 233.24 Infectiology Antibacterial 15A06 Fleroxacin 369.35 Infectiology Antibacterial 15A07 Clavulanate potassium salt 237.26 Infectiology Antibacterial 15A08 Valproic acid 144.22 Central Nervous Anticonvulsant System 15A09 Mepivacaine hydrochloride 282.82 Neuromuscular Local Anaesthetic 15A10 Rifaximin 785.90 Infectiology Antibacterial 15A11 Estradiol Valerate 356.51 Endocrinology Contraceptive 15B02 Acetylcysteine 163.20 Metabolism Mucolytic 15B03 Melengestrol acetate 396.53 Endocrinology 15B04 Bromhexine hydrochloride 412.60 Respiratory Expectorant 15B05 Anethole-trithione 240.37 Metabolism Choleretic 15B06 Amcinonide 502.59 Metabolism Anti-inflammatory 15B07 Caffeine 194.19 Central Nervous CNS Stimulant System 15B08 Carvedilol 406.49 Cardiovascular Antihypertensive 15B09 Methenamine 140.19 Infectiology Antibacterial 15B10 Phentermine hydrochloride 185.70 Central Nervous System 15B11 Diclazuril 407.65 Metabolism 15C02 Famciclovir 321.34 Infectiology Antiviral 15C03 Dopamine hydrochloride 189.64 Cardiovascular Antihypertensive 15C04 Cefdinir 395.42 Infectiology Antibacterial 15C05 Carprofen 273.72 Metabolism Anti-inflammatory 15C06 Celecoxib 381.38 Metabolism Anti-inflammatory 15C07 Candesartan 440.47 Cardiovascular Antihypertensive 15C08 Fludarabine 285.24 Oncology Antineoplastic 15C09 Cladribine 285.69 Oncology Antineoplastic 15C10 Vardenafil 488.61 Cardiovascular Erectile dysfunction treatment 15C11 Fluconazole 306.28 Metabolism Antifungal 15D02 5-fluorouracil 130.08 Oncology Antineoplastic 15D03 Mesna 164.18 Oncology Chemoprotectant 15D04 Mitotane 320.05 Endocrinology Antineoplastic 15D05 Ambrisentan 378.43 Cardiovascular Antihypertensive 15D06 Triclosan 289.55 Infectiology Antibacterial 15D07 Enoxacin 320.33 Infectiology Antibacterial 15D08 Olopatadine hydrochloride 373.88 Allergology Antihistaminic 15D09 Granisetron 312.42 Endocrinology Antiemetic 15D10 Anthralin 226.23 Dermatology Antipsoriatic 15D11 Lamotrigine 256.10 Central Nervous Anticonvulsant System 15E02 Clofibrate 242.70 Metabolism Antilipemic 15E03 Cyclophosphamide 261.09 Immunology Antineoplastic 15E04 Aripiprazole 448.40 Central Nervous Antipsychotic System 15E05 Ethinylestradiol 296.41 Endocrinology Contraceptive 15E06 Fluocinolone acetonide 452.50 Metabolism Anti-inflammatory 15E07 Sparfloxacin 392.41 Infectiology Antibacterial 15E08 Desloratadine 310.83 Allergology Antihistaminic 15E09 Clarithromycin 747.97 Infectiology Antibacterial

249 15E10 Tripelennamine 291.83 Allergology Antihistaminic hydrochloride 15E11 Tulobuterol 227.74 Respiratory Bronchodilator 15F02 Topotecan 421.46 Oncology Antineoplastic 15F03 Atorvastatin 558.66 Metabolism 15F04 Azithromycin 749.00 Infectiology Antibacterial 15F05 Ibudilast 230.31 Metabolism Anti-inflammatory 15F06 Losartan 422.92 Cardiovascular Antihypertensive 15F07 Benztropine mesylate 403.54 Central Nervous Antiparkinsonian System 15F08 Vecuronium bromide 637.75 Metabolism Muscle relaxant 15F09 Telmisartan 514.63 Cardiovascular Antihypertensive 15F10 Nalmefene hydrochloride 375.90 Central Nervous System 15F11 Bifonazole 310.40 Infectiology Antifungal 15G02 Gatifloxacin 375.40 Infectiology Antibacterial 15G03 Bosentan 551.63 Cardiovascular Vasodilator 15G04 Gemcitabine 263.20 Oncology Antineoplastic 15G05 Olmesartan 558.60 Cardiovascular Antihypertensive 15G06 Racepinephrine 219.67 Cardiovascular Bronchodilator hydrochloride 15G07 Montelukast 586.20 Respiratory Antiasthmatic 15G08 Docetaxel 807.90 Oncology Antineoplastic 15G09 Cilnidipine 492.53 Cardiovascular Antihypertensive 15G10 Imiquimod 240.31 Dermatology Antiviral 15G11 Fosinopril 563.68 Cardiovascular Antihypertensive 15H02 Imatinib 493.62 Oncology Antineoplastic 15H03 Moxifloxacin 401.44 Infectiology Antibacterial 15H04 Formoterol fumarate 804.90 Respiratory Antiasthmatic 15H05 Rufloxacin 363.41 Infectiology Antibacterial 15H06 Pravastatin 424.54 Metabolism Antilipemic 15H07 Rosiglitazone Hydrochloride 393.90 Metabolism Antidiabetic 15H08 Rivastigmine 250.34 Central Nervous System 15H09 Sildenafil 474.59 Cardiovascular Antihypertensive 15H10 Acetylsalicylic acid 180.16 Central Nervous Analgesic System 15H11 Hexachlorophene 406.91 Infectiology Antiseptic 16A02 Nelfinavir mesylate 663.90 Infectiology Antiviral 16A03 Silodosin 495.55 Cardiovascular Antihypertensive 16A04 Trimebutine 387.48 Neuromuscular Antispastic 16A05 Nevirapine 266.31 Infectiology Antiviral 16A06 Doxapram hydrochloride 414.98 Respiratory Analeptic 16A07 Amlexanox 298.30 Allergology Anti-inflammatory 16A08 Amorolfine hydrochloride 353.98 Infectiology Antifungal 16A09 Enrofloxacin 359.40 Infectiology Antibacterial 16A10 Ubenimex 308.38 Oncology Antineoplastic 16A11 Troxipide 294.35 Metabolism Antiulcer 16B02 Ipriflavone 280.33 Metabolism Antiosteoporetic 16B03 Ezetimibe 409.44 Metabolism Hypocholesterolemic 16B04 Rizatriptan benzoate 391.48 Central Nervous Antimigraine System 16B05 Tegaserod maleate 417.47 Gastroenterology Gastroprokinetic

250 16B06 Pantoprazole sodium 405.36 Metabolism Antiulcer 16B07 Tegafur 200.17 Oncology Antineoplastic 16B08 Tolcapone 273.25 Central Nervous Antiparkinsonian System 16B09 Altrenogest 310.44 Endocrinology Progestogen 16B10 Felbamate 238.25 Central Nervous Antiepileptic System 16B11 Estramustine 440.41 Oncology Antineoplastic 16C02 (R)-Duloxetine 333.88 hydrochloride 16C03 Donepezil hydrochloride 415.96 Central Nervous Anti-Alzheimer System 16C04 1,8-Dihydroxyanthraquinone 240.22 Gastroenterology Laxative 16C05 Nitazoxanide 307.29 Infectiology Antiprotozoal 16C06 Nateglinide 317.43 Endocrinology Antidiabetic 16C07 Avobenzone 310.40 Dermatology Cytoprotectant 16C08 Algestone acetophenide 448.61 Endocrinology Contraceptive 16C09 Actarit 193.20 Immunology Anti-inflammatory 16C10 Ethoxzolamide 258.32 Ophthalmology Antiglaucoma 16C11 Azatadine maleate 522.56 Allergology Antihistaminic 16D02 Aminacrine 194.24 Infectiology Antiseptic 16D03 Pidotimod 244.27 Immunology Immunostimulant 16D04 Benidipine hydrochloride 542.04 Cardiovascular Antihypertensive 16D05 Perospirone 426.59 Central Nervous Antipsychotic System 16D06 Cefpiramide 612.65 Infectiology Antibacterial 16D07 Fenoldopam 305.76 Cardiovascular Antihypertensive 16D08 Adapalene 412.53 Dermatology Keratolytic 16D09 Diatrizoic acid dihydrate 649.95 Diagnostic Contrastant 16D10 Dofetilide 441.57 Cardiovascular Antiarrhythmic 16D11 Phenprobamate 179.22 Central Nervous Muscle relaxant System 16E02 Cefuroxime axetil 510.48 Infectiology Antibacterial 16E03 Anagrelide 256.09 Haematology Thrombolytic 16E04 Clopidogrel 321.83 Cardiovascular Antiplatelet 16E05 Benzoxiquine 249.27 Infectiology Antiseptic 16E06 Phenothiazine 199.28 Central Nervous Antipsychotic System 16E07 Enalaprilat dihydrate 384.43 Cardiovascular Antihypertensive 16E08 Pregabalin 159.23 Central Nervous Anticonvulsant System 16E09 Homoveratrylamine 217.70 Cardiovascular Antihypertensive 16E10 Zoledronic acid hydrate 290.11 Oncology Antiosteoporetic 16E11 Cefpodoxime proxetil 557.61 Infectiology Antibacterial 16F02 Irbesartan 428.54 Cardiovascular Antihypertensive 16F03 Indinavir sulfate 711.88 Infectiology Antiviral 16F04 Terbinafine 291.44 Infectiology Antifungal 16F05 Histamine dihydrochloride 184.07 Oncology Antineoplastic 16F06 Rasagiline 171.24 Central Nervous Antiparkinsonian System 16F07 Flumethasone pivalate 494.58 Dermatology Anti-inflammatory 16F08 Lofepramine 418.97 Central Nervous Antidepressant System 16F09 Valdecoxib 314.37 Metabolism Antiarthritic

251 16F10 Besifloxacin hydrochloride 430.31 Ophthalmology Antibacterial 16F11 Ritonavir 720.96 Infectiology Antiviral 16G02 Epirubicin hydrochloride 579.99 Oncology Antineoplastic 16G03 Loteprednol etabonate 466.96 Ophthalmology Anti-inflammatory 16G04 Tolterodine tartrate 475.59 Neuromuscular Muscle relaxant 16G05 Lomerizine hydrochloride 505.01 Central Nervous Antimigraine System 16G06 Ampiroxicam 447.47 Immunology Anti-inflammatory 16G07 Alosetron hydrochloride 330.82 Central Nervous Antidiarrheal System 16G08 Risedronic acid 301.13 Metabolism Antiosteoporetic monohydrate 16G09 Palonosetron hydrochloride 332.88 Central Nervous Antiemetic System 16G10 Oxymetholone 332.49 Endocrinology Anabolic 16G11 Latanoprost 432.61 Ophthalmology Antiglaucoma 16H02 Cisatracurium besylate 1243.51 Neuromuscular Muscle relaxant 16H03 Pemetrexed disodium 471.38 Oncology Antineoplastic 16H04 Raltitrexed 458.50 Oncology Antineoplastic 16H05 Ceftibuten 410.43 Infectiology Antibacterial 16H06 Valsartan 435.53 Cardiovascular Vasodilator 16H07 Milnacipran hydrochloride 282.82 Central Nervous Antidepressant System 16H08 Triclabendazole 359.66 Infectiology Antihelmintic 16H09 Brimonidine L-Tartrate 442.23 Ophthalmology Antiglaucoma 16H10 Desonide 416.52 Dermatology Antipsoriatic 16H11 Cefprozil 389.43 Infectiology Antibacterial

252